MATERIALS & METHODS

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SW13 cells were cultured in P35G-1.5-7-C-Grid cell locate culture dishes (MatTek Corporation, Ashland, MA) in complete DMEM medium for 1 day after plating.

Immunocytochemistry was performed to measure HNE formation
in cultured spinal cord neurons exposed to arachidonic
acid according to the procedure described earlier (Herbst et al.,
1999). In brief, cultures of spinal cord neurons were prepared
on glass

Glass-bottomed microwell dishes (35 mm, coated with poly- -lysine) were from MatTek Corporation (Ashland, MA, USA). … For imaging studies, cells were plated on to 35 mm poly- -lysine coated glass-bottomed microwell dishes 24–72 h before recording.

To assess steady state cellular distributions of PTHrP, 293 cells were plated in 35-mm glass bottom culture dishes (MatTek, Ashland, MA) and transfected with GFP3-PTHrP chimeras.

Succinimidyl ester of H2DCFDA
was obtained from Molecular Probes (Eugene, OR, USA).
All other reagents were of the highest purity grade available.
A stock solution of H2DCFDA was prepared in dimethyl
sulfoxide (DMSO) and stored at -20°C. HS-Os-1 cells
(1x

RBL-2H3 cells were
grown in minimal essential medium (MEM-a) supplemented with 20% fetal
bovine serum and 100 units of penicillin-streptomycin. The 7mmgrid dishes were used for
the experiments using the BrdU incorporation assay. The 14 mm dishes were

The lungs were then placed in matTek dishes (MatTek Corporation, Ashland, MA) with 1 ml of L15 medium (Life Technologies, Inc.) to keep them moist.

"The typically heavier TNCs were allowed to sediment. TNCs obtained by this process were cultured for RNA extraction or, alternatively, plated in MatTek (Ashland, MA) imaging dishes for calcium wave experiments."

Cells collected into needles were
extruded into a poly-L-lysine–coated MatTek dish (MatTek Corp., Ashland,
MA) containing 20 L of 10% paraformaldehyde and fixed for 30 minutes.
Nonspecific binding was blocked with 100 L Tris-buffered saline (TBS) wit

Neurons were plated at 100,000 per 35mm glass-bottom microwell dish (MatTek).

Candida albicans was grown in 5 ml of YNBNP medium incubated
in a rollerdrum overnight at 378C. The morphological
state of C. albicans was determined microscopically using a
100¥ objective lens on a Nikon Eclipse TE300 inverted microscope
equipped wit

Freshly isolated salivary gland cells were loaded with fura 2 for 45-60 min at
30 °C and allowed to attach to glass bottom
dish (Matek Corporation).

For retina-chiasm cocultures, retinal explants were plated onto 12 mm glass coverslip-bottom dishes (MatTek) coated sequentially
with 0.01% polyornithine (Sigma) and 10 mg/ml (E14.5) or 20 mg/ml
(E17.5) mouse laminin (Invitrogen) for 2 hr each at 37ºC.

valve, draining the mantle fluid, and withdrawing hemolymph
from the adductor muscle sinus using a lo-ml syringe
equipped with a 22-gauge, 1.5-in. needle. Hemolymph
from two or three oysters was expelled slowly into a glass
test tube and kept on ice t

PtK2 cells were grown on 35-mm glass-bottom dishes (MatTek, Ashland, MA) as described by Ayoob et al. [2000b].

Tanycytes were seeded in cloning cylinders positioned on 37 mm culture chambers (MatTek Corp., Ashland, MA) containing poly-L-ornithine- and laminin-coated glass coverslips, as described above. After removal of the cylinder, the cells were maintained

NIH-3T3 cells were plated subconfluently on fibronectin-coated glass
dishes (Mat Tek) 16 hr before mincroinjection. Expression plasmids were
injected into the nucleus by use of an Automated Microinjection System
(Carl Zeiss). After 16 hr, cells were fi

COS-7 cells transfected with either OBRs–YFP or OB-Rl–YFP expression plasmids were grown on 35-mm glass-bottomed microwell dishes (Plastek Cultureware, MatTek Corp., Ashland, MA).

Pol-Scope imaging for metaphase spindles were carried out as
described previously (Liu et al., 2000). Oocytes were imaged using
a Zeiss Axiovert 100 inverted microscope, equipped with a Cohu
analogue video camera and Pol-Scope hardware consisting of li

Stably transfected Balb 3T3 fibroblasts
(expressing high levels of GRPr constructs) were plated in MatTek
glass-bottomed 35-mm dishes at 150 to 200,000 cells/dish. The next
day, each dish was transfected with 0.1 to 1 g of EGFP-N1--
arrestin1 (arr2)

MatTek dishes were purchased from MatTek Corporation (Ashland, MA).

Myc-GRPr cells were plated in glass bottom 35-mm dishes (Mat-
Tek, Ashland, MA) at 150,000 cells/dish and cultured overnight.
Cells were transfected with 0.3 g of pEGFP-N1-arrestin2 or arrestin3-
GFP by LipofectAMINE method following the manufacturer’

"... and seeded (2 ml per dish; density = about 1.2 million cells/ml) into poly-D-lysinecoated 35-mm glass-bottomed dishes (MatTek, Ashland, MA)."

Ventral mesencephalic neuronal cultures were prepared as described
previously (Nashmi et al., 2003). Brain tissue containing primary mesencephalic
neurons of the ventral tegmental area from E14 BDF1 mouse
embryos (Shimoda et al., 1992) was incubated in

Prior to the live observations the parthenogenotes and the fertilized embryos were placed in a glass-bottom chamber (MatTek Corp., Ashland, MA) filled with culture medium and overlaid with pre-warmed mineral oil.

For live cell analysis, cells spread on glass-coated 35 mm dishes (Mattek) were transfected with DNA as described above, and then examined without fixation.

Cells plated on MatTek 35-mm dishes (part no. P35G-
1.5–10-C, MatTek Corp., Ashland, MA, USA) were rinsed
in warm serum-free DMEM (SFM) and placed in SFM for
30 min in a 37 C incubator to chase out cell surface-bound
Tf. Cells were rinsed in ice cold

Stock solutions made in embryo medium and 50 nl of various concentrations were injected into the yolk sack of developing wild-type and perplexed embryos at 20 hr postfertilization (hpf). Partial rescue of eye, jaw, and fin development was observed at a mi

IR3-GFP-expressing HEK293 cells were plated on 35 mm glass-bottom sterile tissue
culture dishes (Mattek Co. , Ashland, MA) and transiently transfected with l g Flagtagged
IKK&, TBK1, IKKjJ, and visualized 48 h later by confocal microscopy.

All TIRF experiments were performed on a BON NPY-GFP clone, named BC6,which was selected and plated on uncoated glass-bottom dishes (P50G-1.5-14-F, MatTek Cultureware, Ashland, MA).

Calcium imaging was done in the Vanderbilt University Cell Imaging Resource. A laser scanning confocal microscope (Zeiss LSM410, Seiles Instruments, St. Louis, MO), with diffraction-limited focusing of a laser beam, coupled with a special filter placed "c

Untreated and LT-treated PMNs (1
105 cells in 2 mL of RPMI) were added to 35-mm glass-bottom microwell dishes (MatTek Cultureware) coated with 0.1% fibronectin (Sigma).

Thirty patients who were admitted to Memorial Hermann Hospital
were identified by the obstetrics faculty of the University of Texas
Medical School at Houston. Sixteen patients were diagnosed with
severe preeclampsia on the basis of the definition set b

For the microscopic fluorimetric measurement of intracellular free Ca2+ concentrations ([Ca2+]i), A431 cells were plated on poly-D-lysine coated glass bottom dishes (MatTek, Ashland, USA).

After removing the legs of the filter units, living or fixed cells were observed by placing the entire filter unit on two 50µ m tape spacers attached to the coverslip of a coverslip-bottomed 35 mm dish (Mattek, Ashland, MA) mounted on the stage of an inve

Submandibular glands were removed and placed in ice-cold
external solution with 0.02% soybean trypsin inhibitor and 0.1% bovine
serum albumin. Each gland was cleaned and finely minced. The minced
tissue was transferred to 8 ml of external solution cont

For Hoechst staining after serum removal or staurosporine treatment,
300,000 cells were plated on collagen-coated, glass bottom 35-mm tissue
culture dishes (Mat-Tek, Ashland, MA). After experimental treatments,
cells were rinsed three times with PBS

Cells were seeded on 35-mm glass-bottom dishes (MatTek, Ashland, MA) and microinjected with 1 pg of CFP-CA or
CFP-VCA vector DNA by using a MO-150 micromanipulator and an IM200 pressure injector (Narishige, Tokyo).

Frozen hamster eggs (Charles River Laboratories, Wilmington,
MA) were thawed and transferred to 35-mm
glass-bottom microwells (Mat Tek Corporation, Ashland,
MA) in BWW medium. To remove zona, the washed eggs
were treated with 0.05% trypsin (Irvine Sci

Pregnant Sprague—Dawley rats were narcotized by COµ
inhalation followed by cervical dislocation. E15 embryos were
removed from the uteri by Caesarean section, rapidly decapitated
and placed in phosphate-buffered saline at room temperature
(20—22 °C).

KB cells were plated 1 day in advance on MatTek tissue culture dishes (glass bottom No. 15, uncoated, ç-irradiated, Part # P35G-1.5-10-C) in RPMI 1640 media.

The VSMCs were isolated from a rat cremaster arterioleas previously described.68 Low passage VSMCs weretrypsinized and then centrifuged to form a pellet. The cell pellet was dispersed in cell culture media, and the cells werecultured on glass-botto

CaSki cells transfected with
-arrestin-2-GFP or HEK-293 cells cotransfected with the full-length human P2X7 receptor and
-arrestin-2-GFP were plated on 35-mm glass-bottomed culture dishes (MatTek, Ashland, MA).

Cells were grown in a glass-bottom culture dish (MatTech, USA).
GFP fusion plasmids (1 mg) were transfected into cells with
LipofectaminePlus (Gibco BRL) according to manufacturer’s
methods except that the incubation time with DNA was reduced to
1.5 h

Cultures were grown on glass-bottomed dishes (MatTek, Ashland, MA), maintained in culture for 3 or 4 days or 3 weeks and then fixed for 10 min in 4% paraformaldehyde.

A total of 12 adult Wistar rats were anaesthetised
with pentobarbitone (40 mg/kg ip). The cauda equina
was exposed by dorsal laminectomy and the dorsal
roots (DR) S1, S2, and L1 were completely transected
on the right side of the body approximately 1

After the final precipitation step, neurons were suspended in fresh Dulbecco’s modified Eagle’s medium/F-12 (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and plated at a density of 2.5 x 10^5 cells in

After incubation with the dyes, polymethylmethacrylate strips or silicone elastomer
disks were flipped and placed on a 35-mm-diameter glass-bottom petri
dish (MatTek Corp., Ashland, Mass.). Stained biofilms were observed with a
Zeiss LSM510 confocal sc

HeLa cells transduced with retroviruses encoding the indicators and cultured in glass-bottomed dish
(MatTek) were loaded at room temperature (23 1C–25 1C) with 5 mM indo-5F AM or fura-2 AM in physiological salt solution…

Adipose tissue was first
washed several times with HBSS, minced into small pieces, and
digested with 0.8 g/L type I collagenase in a shaking water bath at
37°C for 30 min. Adipocytes were then filtered through sterile
500-m nylon mesh and cultured in

Cos-1 cells were plated into 35-mm dishes containing
a glass coverslip-covered 15-mm cutout (MatTek Corporation) and transfected
the next day with 500 ng of GFP expression plasmid. Cells were examined
24 h posttransfection with a Zeiss Axiovert epifluo

Cells were seeded on glass-bottomed
coverslip dishes (Matteck Corp., Ashland, OR) 24–48 h prior to injection.
Nuclear microinjection was performed using an automated microinjection
system (Eppendorf Transjector 5246, micromanipulator 5171).
Identical

HEK293A cells were plated at a density of 0.1  106/35-mm glass-bottomed microwell dish (Mat Tek).
The DNA:Opti-MEM ratio was 0.5 g of the indicated constructs (see
figure legends) into 50 l of Opti-MEM (Invitrogen), and the LipofectAMINE:
Opti-MEM r

Cells growing on a glass-bottomed dish (MatTek) or a coverslip were microinjected
with 10 nM siRNA and 0.008


g/


l plasmid CFP (pCFP)-
Golgi as a marker in the first release of a double thymidine block. As for
time-lapse microscopy, different si

Transfection reactions were carried out with LipofectAmine PLUS
(Invitrogen). HeLa cells cultured in a 35 mm glass-bottom culture dish
(MatTek, Ashland, MA, USA) were transfected with 250 ng of
plasmid DNA, and incubated at 37°C for 1.5 hours. For cotr

Isolated epithelia
were then placed on a small spatula and transferred into
fibronectin- or laminin-coated culture wells that contained 50 l of
medium 199 (with Earle’s salts, 2200 mg/ l sodium bicarbonate, 25 mM
HEPES, and 0.69 mM L-glutamine), supp

First, about five drops of 0.1 N NaOH were added to the 35-mm dish (glass bottom, uncoated, no. 0; MatTek, Ashland, MA) and air-dried.

In preparation for experiments, cells were cultured in DMEM with 0.5% FCS for 24 hours, trypsinized (Trypsin-EDTA, Gibco), and then plated (1.5104 cells/dish) onto glass-bottomed 35 mm dishes (MatTek Corp)…

Importin
(mouse Rch1) and  were constructed with pEGFP-C1 plasmid
(CLONTECH Laboratories, Inc.). HeLa cells were plated onto a glass bottom
dish (MatTek Corporation) cultured for 1–2 d before use. After the
constructs were transfected using Effecten

Coverslips for live cell imaging were placed in Rose chambers (9) or grown directly on glass-bottom culture dishes (MatTek, Ashland, MA).

Jurkat T cells and MCF7 adenocarcinoma cells were
obtained from ATCC (Rockville, MD). TNF-a was purchased
from PeproTech (Rocky Hill, NJ); C6-ceramide
was purchased from Matreya (Pleasant Gap, PA);
[g-32]ATP was purchased from NEN Life Science Product

An aliquot (0.5 ml) of isolated scallop myofibril suspension was placed into a glass bottom Petri dish (MatTek Corp., Ashland, MA) and the dish was centrifuged at 4000g for 10 min.

HeLa or N/TERT-1 cells expressing GFP–histone 2B were grown on gridded coverglass bottom dishes (MatTek, CatNo. P35G-1.5-7-C-grid) for 26 h (HeLa, 2 £ 105 cells per dish) or 3 d (N/TERT-1, 4 £ 104 cells per dish) before imaging.

OC cultures were prepared from cochleae of Cldn14/,
Cldn14þ/ and Cldn14þ/þ mice at P3–P5 according to the
procedure described (87) with some modifications. The
cochleae were dissected in Leibowitz cell culture medium,
L-15 (Invitrogen, Carlsbad, CA

Freshly isolated B cells (2  106 cells/ml in IMDM supplemented with
2.5% FCS) were incubated with fura 2-AM (1
M for 30 min at room
temperature) and put on poly-L-lysine-coated glass bottom microscopic
dish (MatTek) for 15 min, allowing the cells to

H2DCFDAloaded
cells in a 0.2 ml suspension at a concentration of
1x106 cells/ml were placed onto a glass coverslip at the bottom
of a microwell dish (35 mm dish, poly-D-lysine-coated;
MatTek, Ashland, MA, USA) for 2 min at 20°C.

Silicon molds used for fabrication of the PDMS chips were
realized using DRIE using the Bosch process. Molds with two
different etch depths, 15 and 25 mm (Veeco Dektak 8 surface
profiler), were fabricated. In Fig. 2 the three-dimensional
structure of

HeLa cells were
plated in 12 well plates, 105 cells per well, 24 h prior to
infection. The vaccinia stock (20 íL of approximately 109
plaque forming units/mL) was mixed with 30 íL of 0.25
mg/mL trypsin solution in Dulbecco’s phosphate-buffered
saline

Cortical precursors from green fluorescent protein (GFP; Okabe et al.,
1997) / and / mice were seeded on poly-L-lysine- and laminin-coated glass coverslips to obtain 25% of GFP cells. This low proportion
of GFP cells makes it possible to reliably

Calcium imaging in single PC neurons was done in a dissociated culture
of PC neurons (Rhines et al., 1993). The PC was desheathed and
isolated in Mg2 buffer, digested with 1% protease (type IX; Sigma) at
34°C for 1 hr, and dissociated by trituration.

3T3-L1 adipocytes, expressing appropriate cDNA constructs, were seeded in glass-bottomed dishes (MatTek Corporation).

MC were cultured to confluence in special glass-bottom microwell dishes (MatTek Corporation, Ashland, MA) and then loaded with fura-2 by incubation with 5 µmol/L Fura-2 AM in Hanks balanced salt solution (HBSS) containing 2.0 mmol/L CaCl2 and 1 mmol/L MgC

To examine the diapedesis of monocytes, EA.hy 926 cells were cultured on Matrigel on T-type dishes (MatTek, Ashland, USA), each containing a glass coverslip as the bottom surface.

u l b e c c o ’s modified Eagle’s medium (DMEM) was purchased
from Nissui Pharmaceutical (Tokyo, Japan); fetal bovine serum (FBS), from
JRH Biosciences (Lenexa, KS); [ -3 2P ] AT P, [ -3 2P ] d C T P, D- [ 5 -3H]glucose, and
[3H ] w a t e r, from Du Po

For time-lapse analysis, adherent cells were grown in 35mm glassbottomed
microwell dishes (MatTek), and treated in 3ml of phenol-red free
DMEM (Gibco) supplemented with 10% FCS, 20mM HEPES (pH 7.3),
2mM L-glutamine, 200 mg/ml penicillin, 100 mg/ml stre

T. gondii tachyzoites (strain RH) were
cultivated in human foreskin fibroblast cells as previously described
[59]. Parasites were harvested soon after emerging from host cells,
passed through a 3-lm filter (Whatman #110612, Whatman, Brentford,
Middles

"After reaching confluence, cells were plated on 35 mm glass-bottom microwell dishes (MatTek, Ashand, MA, USA) for microinjection" … "NEB1 keratinocytes and NIH 3T3 fibroblasts were seeded in MatTek dishes and allowed 3 days to grow to near con-fluency. C

As mentioned previously, cells were grown on the underside of
Millipore filter units. After removing the legs of the filter units, living
or fixed cells were observed by placing the entire filter unit on two
50-mm tape spacers attached to the coverslip

Briefly, glass-bottomed culture dishes (MatTek, Ashland, MA) were coated with collagen using 0.03% collagen in 0.1 N acetic acid.

To label lysosomes, day 4–5 immature dendritic cells were…plated for 30min on poly-L-lysine pre-coated number 1.5 coverslips attached to 35-mm dishes (MatTek).

ER293 cells were obtained from Stratagene (Heidelberg, Germany) and
grown in an 8.5% CO2-humidified atmosphere at 37C in 88% (v/v) phenolred
free Dulbecco’s modified Eagle’s medium (GibcoBRL, Karlsruhe,
Germany) containing 10% (v/v) Mycoplex Fetal Cal

Stained catheter segments were transferred to a glass-bottom petri dish (coverslip
1.5, 35-mm disk P325G 1.5-14C; MatTek, Ashland, Mass.) in an on-end
orientation. Biofilms were observed with a Nipkow disk-based confocal microscope
(Axiovert 200; Zeiss

Cells were seeded onto glass coverslips in 24-well plates in conjugation
medium (RPMI medium without phenol red/0.5 % FCS) and allowed
to attach for 20 min at 37ºC. T cells were treated with either
200 nM phorbol-12,13-dibutyrate (PdBu, Calbiochem) or

To examine the localization of GFP fusion
proteins, axenic cells were incubated overnight in HL5 medium supplemented
with 10 microg/ml G418 in 35-mm glass bottom microwell dishes
(MatTek Corp.).

Cells were grown in glass-bottomed microwell
dishes (Mat Tek, MA) for 40–48 h to 50% confluence.
The standard FLIP experiment in Figure 1
was performed as described by Kimura et al.
(2002) using a Radiance 2000 confocal microscope
(488-nm-laser line;

Skeletal myoblasts were isolated from the breast
muscles of 9-day-old quail embryos and plated on collagen-
coated 35-mm MatTek (Ashland, MA) dishes at
concentrations of 105 cells per dish according to procedures
described in Dabiri et al. [1999]. Fol

Adult flies were dissected in Schneider’s Drosophila
medium (Invitrogen). For vital imaging of V-ATPase
expression with the vhaSFD::GFP gene trap line (5),
tubules were allowed to adhere lightly to a poly-Llysine
treated coverslip glued to a window in the

One day after transfection, cells were divided into collagen-coated 35-mm glass bottom dishes (MatTek, Ashland, MA)
for confocal microscopy.

Microscopy of fixed and
living cells expressing GFP fusion proteins was performed as described extensively
in reference 23. For GFP analysis in living cells, cells were seeded in
glass-bottomed petri dishes (Mattek Corp., Ashland, Mass.) transfected as

After stained cells were transferred onto poly-d-Lyscoated dishes (Glass Bottom Microwell Dishes, MatTek Corporation, Ashland, MA), they were placed on the inverted platform of a confocal laser scanning microscope (Meridian Instruments Far East, Tokyo).

The pSPT-cfos was treated with EcoRI to cut off c-fos DNA (2.1 kb), and
the c-fos DNA fragment was inserted into the EcoRI site on pME18S
expression vector driven by SRa promoter (pME18S-cfos).
Cos7 cells were cultured with Dulbecco’s modified Eagle’s

To obtain real-time images of bacteria expressing GFP-labelled
proteins interacting with host cells, either HCT-8 or CaCo-2 cells
were grown overnight at low density on glass bottom culture
dishes (MatTek, Ashland, MA). Strain jf1128 was grown overnigh

Groups of subconfluent MDCK cells cultured on 50 mm MatTek
glass-bottomed Petri dishes were microinjected in the nucleus with
HA-Dvl-2-EGFP plasmid diluted to 25 g/ml in PBS. After a 6 hour
expression period, the HA-Dvl-2-EGFP expressing cells were im

Cells were seeded into 35 mm glass-bottom Petri dishes (MatTek Inc., Amherst, MA) at a density of 2.5 × 104/ml, and allowed to recover for 2 days prior to use.

For time-lapse fluorescence microscopy of living cells,
epifluorescence using inverse optics (Olympus, Hamburg,
Germany) was recorded with an IMAGO slow
scan CCD camera (TILL Photonics, Gra¨ felfing, Germany)
as described previously (Windoffer et al., 200

For time lapse microscopy, cells were seeded on glass-bottom culture dishes (MatTek Corp., Ashland, MA), grown for 8-48 h after transfection, and incubated …

Primary antibodies were: rabbit anti-EB1 (Rogers et al., 2002)
(1:500), rabbit anti-CLIP190 (Lantz and Miller, 1998) (1:1000), rabbit
anti-b-galactosidase (Sigma), rat anti-tubulin (Serotec), rat mAb anticadherin
(1:5) (Oda et al., 1994), mouse mAb ant

Cells were transiently transfected on 0.18-mm-thick glass-bottomed dishes (MatTek, USA) precoated with 0.5 mg/mL poly(L-lysine) (Sigma).

Briefly, hippocampi from postnatal day 2 Sprague-Dawley rat pups were enzymatically and mechanically dissociated and plated into poly-lysine-coated glass-bottom petri dishes (Mattek).

For live cell recordings, cells were sub-cultured at least 2 days before
each experiment into 35 mm dishes which had been modified with a
hole in the bottom to which a #1 thickness glass coverslip was glued
(Mat Tek Corp, Ashland, MA). The dishes were

For confocal microscopy, cells were plated onto 35-mm Mattek dishes (Mattek, USA) at 1.6×105 cells in 2 ml of medium. HeLa cells (ECACC No. 93021013) were grown in MEM with Earle’s salts, plus 10% FCS, and 1% NEAA at 37°C, 5% CO2….

Briefly, six
mutations were created in which each codon for nonvaline residues within
a region spanning Lys231–Met239 was converted to a valine codon. The
mutated GP Iba cDNAs were then transfected into Chinese hamster ovary (CHO) bIX cells (which stab

RAW 264.7 cells were plated
48 h before use on 35-mm glass-bottomed microwell dishes
(MatTek Corp.) with 20 U/ml IFNg
, and OVA was added to
10 mg/ml for the final 16 h. DO11-GFP cells were added to the
dish at fivefold excess, centrifuged onto the R

Glass bottom 35-mm cell culture dishes (MatTek, Ashland, MA) were precoated overnight at 4 °C with antibodies to CD3 and
CD28 or LFA-1 and diluted to 10 g/ml in phosphate-buffered saline.

Suspended cells were washed twice and re-suspended in Dulbecco’s
minimum essential medium without riboflavin and phenol red
(Biological industries, Beit Haemek, Israel) supplemented with
30mM HEPES (Sigma) and 15 or 0.5% fetal calf serum. The cells
we

For immunofluorescence, the cells were plated onto 12 mm glass coverslips prior to transfection, while for TIR-FM experiments cells were plated onto glass-bottom 35-mm tissue culture dishes (MatTek, Ashland, MA).

Wildtype cells carrying plasmid with the GFP-tagged sad11 gene were
exponentially grown to 3 3 106/ml in 20 ml minimal EMM2 medium. One or 2 ml of the culture were centrifuged at 10,000 rpm for 1 min and resuspended in 80–100 ml Edinburgh minimal medium

Cells were grown on solid YES medium at 33C. To induce meiosis, the cells were transferred to solid ME medium and incubated at 26C for about 12 hr. They were then suspended in EMM-N medium supplemented with appropriate amino acids for live observations.

RAW-TT10 cells were transfected with
dyn K44A -pTIGZ and plated on 35-mm glass-bottomed microwell dishes (MatTek Corp.). 18 h later, the dish was mounted on a Zeiss Axiophot microscope equipped with a cooled CCD camera (Princeton Instruments) and Me

Infected HFKs (105) were grown on 35-mm-diameter glass-bottomed dishes (MatTek Corporation) coated with type 1 collagen (Sigma).

After the electroporation,
the cells were collected by centrifugation, resuspended in
DMEM, and plated on glass-bottom microwell dishes
(MatTek, Ashlend, MA)

The cells were divided into 35-mm glass-bottom dishes (MatTek Corporation) and then were transfected with 0.5 mug of the pEGFP-AR and 2 mug of the pTriARN34, or 2 mug of the empty vector…

Cow tracheae were obtained from a slaughterhouse,
and tracheal epithelial cells were isolated by protease
as previously described (15). Briefly, strips of epithelium were
pulled off the submucosa, washed four times with phosphatebuffered
saline (PBS)

Lipopolysaccharide (LPS) from Es che richia co li ce ll
wall, penicillin, streptomyc in, HEPES, metrizamide,
leupeptin, pepstatin A, aprotinin, phenylmethylsulfonyl
fluoride (PMSF) and thiazolyl blue dye (MTT) were
purchas ed from Sigma Chemic al Co.

Human coronary artery smooth muscle cells, which
were certified to be a virus-free and a pure cell population
on the basis of staining patterns for
-actin, were
obtained from Cell Application, Inc. (San Diego, CA).
The cells were routinely maintained

For time-lapse laser scanning microscopy, cells were seeded
at a density of 2
105 cells per 3.5-cm in collagen I-coated
plates with a glass bottom which were purchased from MatTek
Corporation (Ashland, MA). Uncoated plates were coated for
4hat 378Cwit

Cover glass-bottomed culture dishes coated with poly-D-lysine (#P35GC-0-14-C) were from MatTek (Ashland, MA). AND
Once loaded with fluo-3, the cells were diluted 1:9 with RPMI 1640, incubated for an additional 30 min, washed with Tyrode-HEPES buffer (20

Preadipocytes were plated in 35-mm dishes (P35G-0-14-C, MatTek).

COS-7 cells were plated onto glass-bottom tissue culture plates (MatTek, Ashland, MA).

A7r5 smooth muscle cells (SMC) were
maintained in polystyrene flasks between passages 2 and 15,
and cultured in media containing DMEM, supplemented with
10% of fetal bovine serum and antibiotics. Cells were passed
every 3 days, when reaching 80% conf

...this study we used ECV304 cells (European Collection of Animal Cell Cultures, Salisbury, UK) grown on glass bottom microwells (MatTek Corporation, Ashwood, MA, USA). Cultures were maintained in Medium-199 (Gibco, Merelbeke, Belgium) with 10% fetal bovi

Kinase deficient mouse EphB2 was
generated by site directed mutagenesis leading to exchange of Lys 660 to Arg. To generate
EphB2–YFP (pJK12), EYFP was cloned in frame into the juxtamembrane
region of Flag–EphB2. NIH3T3 cells stably expressing full-leng

Changes in the pHi of
RGM1 cells were detected with an image analyzer (Attofluor Ratio-
Vision, Karl Zeiss Co., Ltd., Obernkochen, Germany). RGM1 cells
cultured in a glass-bottomed culture dish (MatTek Co., Ashland, MA)
were incubated with 0.3 mM BCEC

Cells cultured on glass-bottomed petri dishes (P35GC-101; MatTek Co., Ashland, MA) were washed with Hepes-buffered HBSS and loaded with 10 mM fura2-AM for 1 h.

Osteoblasts were plated on a glass-bottomed dish (Mat-Tech) and loaded by
exposure to 10µM Fluo3/AM (Wako Chemicals), a fluorescence indicator of Ca2+, at 37 Co
for 2 hours. After being rinsed with phosphate-buffered saline (PBS), the dish was mounted

Transiently transfected cells plated on poly-d-lysine-coated glass bottomed 35 mm micro-well dishes (MatTEK, MA, USA) were loaded with 2 M Fura-2- acetoxymethylester (AM) diluted in growth medium, at 37 °C for 30 min.

Embryonic heart cells are grown
directly on 35 mm microwell dishes with 1.5 mm
glass bottom (MatTek, Ashland, MA). The quail
skeletal myoblasts are grown on these dishes
after they have been coated with rat tail collagen
(Collaborative Biomedical Pro

Neocortices from day 15 murine embryos were dissociated and
plated on confluent astrocyte cultures at 1 week in vitro, resulting
in mixed astrocyte-neuronal cultures [18]. Preparation of pure neuronal
cultures were generated by plating dissociated neoc

For pHi measurements, MCs were
plated on 35-mm petri dishes containing a glass coverslip
bottom (MatTec, Ashland, MA) and were used 5–7 days later.
Cells were incubated in 0.5% FBS-containing RPMI 1640 for
24 h before use. Before each experiment, the

Mouse fibroblasts (3T3-Swiss albino, ATCC number
CCL-92) were grown asynchronously at 37°C and 5% CO2
in the bottom-glass dishes (Mat Tek, Ashland, MA) containing
Dulbecco’s modified Eagle’s medium with 10% calf
serum.

Caco-2 cells were plated on 35 mm glass bottom dishes (MatTek, Ashland, MA) for 2 days.

Dissociated neurons from 8–16 week-old SCG were plated at a density of one-half ganglion per one 35 mm poly-l-lysine-coated glass bottom petri dish (no. 1.5, MatTek Corp.) and incubated at 37 °C in a 5% CO2 environment in MEM.

Confocal analysis was performed to localize DPPIV-like activity
in living Jurkat and transfected Jurkat cells on a Leica
SP
2
AOBS confocal microscope (Leica Microsystems; Mannheim,
Germany). In case of the use of [Ala-Pro]
2
-cresyl violet,
fluor

COS-7 cells were plated on glass bottom 35 mm tissue culture dishes (MatTek Corp., Ashland, MA), and transiently transfected using GenePorter transfection reagent (Gene Therapy System, San Diego, CA) with 2 µg of wild type or mutant hGnRHR cDNA constructs

“The seedlings were transferred to single chambered cover glasses (Labtek II form Nunc plastic ware or Micro well dishes form Mattek. U.S.A.) for microscopic observation.”

Hippocampal neurons from embryonic rats (E18) were obtained
according to the method previously described (Banker and
Cowan, 1977). In brief, hippocampal tissues from 18 d fetal rats were
dissected and treated with 0.25% trypsin and 0.25 mg/ml DNase (Si

Mouse fibroblasts (3T3 Swiss albino, ATCC number CCL-92) were grown at 37 Celcius and a 5% CO2 atmosphere in galssbottomed disehs (MatTek Corp., Ashland, MA) containing Dulbecco's modified Eagle's medium with 10% calf serum.

DC (2  106/ml) were cultured on sterile glass coverslips in 24-well plates
with or without live or HK tachyzoites, or soluble extracts, for 18 h. Cells
were fixed in 4% paraformaldehyde, permeabilized in saponin buffer, and
stained with biotin-conjuga

Cells cultured on poly-lysine coated glass-bottom dish (35 mm, MatTek Co., Ashland, MA, USA).

For Fig. 7, NRK-GFP-GBF1 cells were grown on Plastek Cultureware glass bottom
microwell dishes (MatTek Corp) and imaged on the temperature-controlled stage of a Zeiss Axiovert 200M microscope equipped with an UltraVIEW ERS 3E spinning disk.

Cells were collected from MMTV-PyMT mice. The needle
contents were extruded into a 500 µl eppendorf tube with
PBS/BSA/EDTA and diluted to 100 µl in the same buffer.
10 µl was removed and stained with DAPI to get an
approximate cell count. The other 90 µl

Porcine kidney proximal tubule cells
(LLC-PK1, ATCC, Rockville, MD) were grown on 18-mm-diameter coverslips (Fisher Scientific, Itasca, IL) for quantitative
uptake experiments, or 35-mm-diameter cell culture
dishes with a grided coverslip attached to t

Five hours after transfection, cells were split into four 35-mm glass bottom culture dishes (MatTek).

For these experiments, we
generated sequence-verified clones for ten representative gene products as N-terminal fusions to a green fluorescent marker protein (GFP) and examined their corresponding
localization patterns in transient transfections
of HEK

T. brucei cells were plated on the surface of agarose gels prepared in cultivation
medium and air dried briefly to limit cell motility. The gel was
placed in an imaging dish (MatTek) with the cell side facing the coverslip.
Cells were imaged at 28C in

For confocal imaging studies, the cell suspension was diluted with MEM to a final concentration of ~5×105 cells/ml and 1 ml added to poly-L-lysine-coated (1 mg/ml) glass-bottom culture dishes (35 mm; MatTek, Ashland, MA, USA) containing 1 ml MEM with 10%

IMLF/ or SkMc cells were plated on 35 mm glass-bottomed
MatTek plates at 1.25  104 cells/cm2 and infected with AdcPLA2

as described above. The cells were rinsed once with PBS
and incubated for 15 min in ice-cold fixative containing 3.2%
paraforma

Fura-loaded oocytes were washed extensively with KSOM and transferred to poly-D-lysine-coated glass-bottom dishes (MatTek Corp., Ashland, MA).

Cells were cultured on 35 mm glass bottom microwell dishes (Mattek Corp. Ashland, MA) at a density of 1000 cells/dish. Intracellular total caspase activity was measured in single cells using the fluorophore tagged pancaspase inhibitor, carboxyfluorescein-

Cells for imaging were subcultured onto 25-mm glass
coverslips and then placed into a watertight cell imaging chamber
at room temperature, or were subcultured onto 35-mm culture
dishes with integral no. 0 glass coverslip bottoms (Mattek).

COS-7 or HeLa cells were plated at 2.5  105
cells/3-cm glass bottomed dish (Mattek) 6 h prior to transfection. Cells
were then transfected using FuGENE (Roche Applied Science) with a
1:1 ratio of target DNA to YFP-mito (Clontech) and a total of 1
g

HeLa cells (ATCC: CCL-2) were cultured on glass coverslips for fixed cell analysis and on MatTek glass bottom dishes (MatTek, Ashland, MA) for in vivo imaging in DMEM for 1 day.

Transfection of cells for confocal experiments was performed in 35 mm glass-bottom dishes (MatTek, Ashland, MA). Briefly, 5  105 cells were seeded into each dish and incubated overnight.

[Ca2
]i was measured as described previously.10 Briefly, endothelial cells were grown to confluence in 35-mm glass-bottom dishes (MatTek). The cells were incubated at 37°C for 10 minutes with 1mol/L fura-2/acetoxymethyl ester and then washed with the

A549-cells were seeded in 35-mm glass bottom dishes (MatTek Corp.) overnight. Cells were transfected with GFP-huTLR9 using Polyfect (Qiagen) according to the manufacturer's instructions or incubated in medium.

Approximately 50–150 neurons were plated on poly-L-lysine (Sigma Chemical Co., St. Louis, MO) coated culture
dishes (P50G-0-14-F) containing L15 media with filtered hemolymph (1:1). Pleural ganglia were also organotypically cultured on the culture dishes

The resulting cells were split
and plated on Mattek dishes covered with Matrigel (1:5) in the presence or
absence of EGF (1 nmol/L) for 4 hours at 37°C. The cells were then lysed
directly on the dish for total RNA extraction.

For time-lapse fluorescence microscopy of living cells, phenol-red–free
Hanks’ medium (Invitrogen GmbH) was used. Images were recorded by
epifluorescence microscopy using inverse optics (Olympus, Hamburg, Germany)
and an attached IMAGO slow-scan CCD ca

For live cell microscopy, cells were seeded on 35 mm glass bottom dishes (MatTek Corporation), and transfected with the indicated plamids the following day.

COS1 cells were grown on poly-D-lysine coated coverslips (MatTek, Ashland, MA) in DMEM supplemented with 10% fetal calf serum. COS1 cells were transfected with CKAR-pcDNA3 using the Effectene kit (Qiagen, Valencia, CA)."""

Cells were seeded onto a collagen-coated
glass-bottomed 3.5-cm dish (MatTek Corp., Ashland, MA) and serumstarved
for 18 h. Following stimulation with DMEM at pH 6.4 and 8.1
containing 0.1% BSA for 15 min at 37 °C, the cells were fixed with PBS containi

Pregnant mice were anesthetized terminally with an intraperitoneal
injection of ketamine–xylazine (80:20 mg/kg). Embryos were
removed from the uterus, washed in ice-cold Hanks balanced salt
solution (HBSS), and placed in ice-cold Roswell Park Memorial

The Plasmodium berghei NK65 strain transformed to express
GFP at the sporozoite stage was described previously (Natarajan
et al., 2001). Parasite stocks were refreshed after several months
by natural transmission from mosquitoes to mice. Mice developin

Cells were plated 24-48 hours prior to injection on live cell dishes (MatTek, Ashland, MA, USA)

Bovine serum albumin (BSA; low endotoxin) was purchased from Sigma (St. Louis, MO, USA), and phosphate-buffered saline (PBS), Dulbecco’s modified Eagle medium (DMEM), and RPMI 1640 culture medium were purchased
from NISSUI Pharmaceutical Ltd. (Tokyo, Jap

Single islet cells were suspended in RPMI 1640 medium (GIBCO/
Invitrogen) supplemented with 10% FBS, 11 mM glucose, 100 IU/ml
penicillin, and 100 g/ml streptomycin and allowed to attach to
poly-L-lysine-coated gridded glass-bottom culture dishes (MatT

For microinjections Saos-2 cells were cultured on glass-bottomed
microinjection dishes (MatTek Corporation) or in 35-mm dishes containing
glass Cellocate coverslips (Eppendorf). The cells were microinjected
in the nucleus with DNA (50 ng/
l in phosphat

HeLa cells or Madin±Darby bovine kidney (MDBK) cells were incubated
in DMEM (Sigma) supplemented with 10% heat-inactivated fetal bovine
serum (FBS) and penicillin/streptomycin (100 IU/ml and 100 mg/ml,
respectively) at 37°C in a 5% CO2 atmosphere. Cult

Adult mouse DRG neurons were isolated as
previously described (33). First, animals were anesthetized by an
overdose of ether and bled from the abdominal aorta. Next, the spinal
cord was isolated and the ganglia dissected using microforceps. After
remo

Oocytes and eggs were broken with fine tungsten needles in buffer A containing 0.01% acridine orange. They were placed in poly-Lysine-coated glass-bottom dishes (MatTek Corp., Ashland, MA) and incubated for 5–10 min at room temperature in the dark.

Fertile white leghorn chick eggs were acquired from a local supplier and incubated at 38°C until approximately the 5-
8 somite stage (ss) of development. Eggs were rinsed with 70%
alcohol and 3 ml of albumin was removed before cutting a window
in the s

For laser confocal
microscopy, cells grown on 35-mm MatTek imaging dishes (Cat no. P35G-0-
14-C, Ashland, MA) were washed with PBS and then were incubated at 37°C
in the presence of 100 nmol/L RGD-Cy5.5 for 1 hour. Afterward, cells were
washed in ice-

Wild-type Ptk2 cells were grown in DMEM supplemented with 10% fetal
bovine serum under standard tissue culture conditions. All cell-culture
media and sera were obtained from Invitrogen (Carlsbad, CA, USA). Ptk2
cells stably expressing tagged tubulin-YF

Laboratory-raised stocks of the freshwater snail,
Lymnaea stagnalis, at 3 to 6 months of age, with a
shell length of 10–25mm, were used. Lymnaea
normal saline contained 51.3mM NaCl, 1.7mM
KCl, 4.1mM CaCl2, 1.5mM MgCl2, and 5.0mM
N-2-hydroxyethylpiper

The cells were transferred 1 day after transfection to 35-mm glass-bottom culture dishes coated with poly-L-lysine (MatTek, Ashland, MA) and grown for 2 days before use.

Oocytes were imaged at 37°C in HKSOM in a plastic Petri dish with a cover glass bottom (MatTek Corp., Ashland, MA) or in a custom-fabricated cover-glass-bottom chamber.

Dihydroethidium (DHE) enters
cells freely. Superoxides oxidize DHE into ethidium or a structurally
similar product, which intercalates into DNA, producing fluorescence
(5). One to 1.2  106 cells were grown overnight on the optical glass
bottom of a d

ER
HA cells were grown on confocal-ready tissue culture
plates (MatTek Corporation, Ashland, MA, USA) and treated
with vehicle or 1 g/mL Dox for 48 h. Cells were subsequently
fixed overnight with 3.7% formaldehyde in PBS at
4°C. A solution of 3.7% fo

Cells were cultured until reaching confluence. After an
additional 24 h, the medium was removed and the cells were
incubated for 24–36 h with peptide-containing medium.
Ten-micromolar BrdU was then added and the cells were
permeabilized and fixed 45 m

This procedure was performed according to Balda et al.
(30). Sphyngomyelin/bovine serum albumin (BSA) complexes
(5 nM/ml) were prepared in P buffer (10nM HEPES,
pH 7.4, 1mM sodium pyruvate, 10mM glucose, 3mM CaCl,
and 145mM NaCl) using Bodipy-FL-sphyn

3T3 cells grown in Dulbecco modified Eagle medium plus 10% fetal bovine serum on 4-cm glass bottom plates (MatTek Corp.)…

Live cell microscopy was performed using an inverted microscope (model
Eclipse TE200 and TE300; Nikon) heated with an airstream incubator
(Nevtek) to 37C, as measured inside the imaging chambers. The microscopes
were equipped with a robotic stage with

OK cells were obtained from the American Type Culture
Collection and maintained in an appropriate medium [20]. For
transfection,OKcells were plated in a 35 mmglass-bottomed dish
(MattekCorp.,Ashland, MA,U.S.A.) at a density of 2×105 cells.
Cultures at sub

A concentration of 2  104 cells/mL of Rat2 cells was seeded on MatTek glass bottom Petri dishes (35 mm) and incubated at 37 °C under 5% CO2 for 24 h.

The C terminus of HERG was used to
probe for interacting proteins expressed from a human heart library
using the yeast two-hybrid technique. We screened 2.2  106 independent clones from a human heart cDNA
library consisting of recombinant plasmids fus

Particle density in the basic biofilm layer and in food vacuoles of sessile ciliates
was estimated by automated analysis of CLSM images. Image acquisition was
performed with a Zeiss (Jena, Germany) 510 confocal laser scanning microscope.
An argon laser

For determination
of pHi, cells were plated on 35-mm microwell
plastic Petri dishes with 18-mm glass coverslips glued to
1-cm diameter holes in the center of each dish (Mattek Corp.,
Ashland, MA). The coverslips were coated with a 1:1 mixture
of coll

Polarized migration of 3T3 fibroblasts was obtained using the scratch-wound model as described previously (DeBiasio et al., 1987). Approximately 1.0
104 cells were seeded into 35-mm glass-bottom dishes coated with poly-d-lysine (MatTek) and pretreate

WIF-B9 cells were cultured in a humidified 7% CO2 incubator at 37°C as
described previously (Sai et al., 1999). Cells were grown in modified F12
supplemented with 5% fetal bovine serum, 10 M hypoxanthine, 40 nM
aminopterin, 1.6 M thymidine, 50 mg/l stre

Islets were isolated from Flk-1wt/wt mice by dissection
of the splenic portion of the pancreas followed by collagenase P digestion
(Roche Molecular Biochemicals, Indianapolis, IN) as previously described
(20). To increase the yield of islets isolated f

Scaffolds were carefully removed from the culture vessel together with the medium and moved to a glass bottom culture dish (MatTek Corporation). The scaffolds were kept immersed in the medium during the process. Co-cultured scaffolds were investigated uti

Cells were plated (0.5 hemisphere per dish) onto glass-bottom 35 mm dishes (Mattek, Ashland, MA) previously coated
with a mixture of poly-D-lysine (0.5 mg/ml) and laminin (4 mg/ml).

General procedures for fixation, immunolabeling, and image collection
using an MRC1000 confocal microscope have been described (Kimura et
al., 1999; Manders et al., 1999). Cells grown on glass-bottomed dishes
(Mat-Tek) in 100 ng/ml Hoechst 33342 (Harag

CHO-K1 cells were transfected to express 5-integrin/eGFP as previously described (28) (provided
by A.R. Horwitz, Univ. of Virginia). Cells were cultured in Dulbecco’s modified Eagle’s medium,
supplemented with 10% fetal bovine serum, 4 mM L-glutamine,

Plate the dissociated cells on a poly-D-lysine and laminin-coated cover glass of a
MatTek culture dish at a density of 20,000 cells and incubate in a 5% CO2 incubator
at 37°C. After 3 h, flood the plates with 1 mL of growth medium. After 3 d
of culturing,

To provide assurance that our observations were not affected by imaging conditions, we used three different methods for time-lapse analysis o fMMTV arrays. First, cells were grown in a Petri dish with a gridded glass bottom (MatTek Corporation). Partic

Human bronchial epithelial cells were a
gift of Dr. Peter Mohler. The cells were grown in Dulbecco’s modified
Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum
(Sigma). Cells were trypsinized, and 60,000 cells were plated on
1.4-mm

HeLa cells seeded on 35-mm glass-bottom dishes (MatTek, Ashland, MA) were transfected with pH2BEYFP
and the indicated siRNA plasmids in a ratio of 1:10.

Time-lapse video microscopy was performed using a Nikon Diaphot
200 inverted microscope equipped with a CCD camera (Hama-matsu Orca). ICAM-1-GFP APCs were split on 35 mm glass-bottom dishes (MatTek Corporation, Ashland, MA). The dish was placed on a 37 C

One day after transfection, cells were transferred to 9.6 cm2 dishes containing glass bottoms (MatTek Corp., 1.5 ·105 cells per dish).

Dissociated hippocampal neuron cultures were prepared from postnatal
2- and 3-day rat pups as described [41]. Neurons were plated
at a density of 15,000–45,000 cells/cm2 onto poly-L-lysine-coated
cell-culture dishes (MatTek). The cultures were maintain

The live cells were observed under an Olympus IMT-2 inverted microscope
using an ´100 plan apochromat objective lens (S-Plan Apo, NA
1.4). The microscope was attached to a real time laser confocal microscope
system ‘Insight Plus-IQ’ (Meridian Instrumen

An individual egg chamber was then mounted on an uncoated #1.5 glass bottom culture dish (MatTek) in 200 µl Schneider's medium. Slight back pressure was applied by laying a 4 mm2 square #1 glass coverslip (Corning) on top of the egg chamber with Dumont #5

An appropriate number of P388D1 cells transiently or stably expressing the PLA2 fusion proteins were placed on a glass-bottomed dish (MetTek, Ashland, MA) and cultured overnight.

Alternatively,
for MatTek dishes and plates, fix the optically transparent base over the magnets with adhesive tape. Cover with 3 ml complete
fresh DMEM medium with 10% FCS and 50 U ml–1 penicillin and 50 mg ml–1 streptomycin. Carefully pipette 1 ml of co

TR was conjugated to gentamicin
as previously described (32). Yeasts were incubated with TR-gentamicin or
lucifer yellow (LY) for the specified period of time under the same growth
conditions as those used for the growth assay. LY accumulates in the ye

MTLn3-GFP and RAW264.7 (LR5) cells were cultured in
a-MEM with 5% fetal bovine serum (FBS). BAC1.2F51.2F5 cells were
cultured in a-MEM with 10% FBS and 36 ng/mL of Human recombinant
CSF-1 (a gift from Chiron Corp., Emeryville, CA). MTLn3-GFP (n = 50,00

GFP-actin-expressing MTLn3 cells were plated on
tissue culture dishes containing a coverslip (MatTek
Corp.) for 24 h and serum starved for approximately 3 h
before EGF stimulation.

Neurons for intracellular Zn21 imaging experiments were prepared similarly, using 35 mm glass-bottom dishes (MatTek, Ashland, MA) coated with poly-D-lysine/
laminin (100:4 ng/ml).

Cells were cultured in DMEM supplemented with
low (0.5%) FCS for 24 h, trypsinized (Trypsin–EDTA, Gibco), collected,
and then seeded (1.5
104 cells/dish) onto glass-bottomed
35mm dishes (MatTek) that were pre-coated with 500 ng/cm2 fibronectin.

For continuous thioflavin T assay measurements, concentrated
stocks of NM stored in either 6 M GuHCl (fluorescently labeled NM
and NM used for Figures 2C, 2F, and 6) or 4 M GuHCl and 4 M urea
(NM used for all other experiments) were diluted at least 2

Drosophila Schneider cell line (S2) was cultured and RNAi was performed
according the methods of Clemens et al. (2000). Templates for in vitro transcription
were generated by PCR using the primers described in Table S1
(available at http://www.jcb.org/

BICR-M1Rk cells transiently expressing Cx43-GFP or stably expressing Cx26-YFP were plated in 35 mm glass-bottom tissue culture dishes for live imaging (MatTek Corporation, MA).

Hippocampal neurons from embryonic rats (E18) were obtained according to the method previously described[25]. In brief, hippocampal tissues from 18-d-old fetal rats were dissected and treated with 0.25% trypsin in Ca2+-Mg2+-free HBSS at 37 oC for 15 min;

Neurons for intracellular Zn2+ imaging experiments were prepared similarly and plated on 35-mm glass-bottom dishes (MatTek, Ashland, MA), coated with poly-D-lysine:laminin (100 ng/ml: 4 ng/ml).

Cells were plated either onto glass bottom dishes (MatTek, Ashland,
MA) or on autoclaved coverslips (Fisher Scientific).

Cells of a murine immortalized melanocyte cell line, melan-a cells
(generous gift of Dorothy C. Bennett, St George’s Hospital Medical
School, London, UK and Katsuhiko Tsukamoto, University of
Yamanashi, Yamanashi, Japan), were cultured on glass-bottom

Mitochondrial membrane potential
was measured using the potentiometric dye Rh123 (Molecular
Probes, Eugene, OR) loaded at a final concentration of 13 M. This
concentration of dye is not harmful to the cells; in studies of isolated

-cells, exposure o

The peel tissues were then washed with distilled water for 10 min and placed in a 35 mm glass-bottomed microwell dish (Mattek Corporation, Ashland, MA, USA) containing 5 ng ml 1 victorin, to prepare for observation.

INS-1 stable transfectants were plated onto 35-mm
glass-bottom culture dishes with grid (MatTek Corp.). After 24 hours, cells
were treated with staurosporine (Sigma-Aldrich), washed with PBS, and
scanned with a manual/motorized reflected-fluorescence s

Resident macrophages were isolated from peritoneal cavities of wild-type mice (C57BL/6) and
gp91-phox-deficient knockout mice by the method of Badwey et al. (1983). Briefly, mice were
killed by cervical dislocation under anesthesia with diethyl ether, a

"VH10 human primary fibroblasts and U2OS human osteosarcoma cells were cultured on 3.5-cm glass bottom petri dishes (Mattek, Ashland, MA) …"

For video imaging experiments [51], T cells (5 × 105 cells/ml) were incubated with 3.5 ? M fura-2/AM (Dojindo Laboratories, Kumamoto) for 45 min at 37 °C, then the cells were washed four times with the medium. To prevent movement of T cells during the

NIH 3T3 and C6 cells were
grown on glass coverslips overnight before fixation and immunofluorescence
analysis. Cells were fixed in freshly made 4%
paraformaldehyde-PBS for 10 min at room temperature, permeabilized
in 0.2% Triton X-100 in PBS for 5 min

Briefly, cells were seeded on 35-mm glass-bottomed plastic dishes (MatTek Corp.), which were marked with a cross to facilitate the localization of injected cells.

For Ca21 imaging studies, cultures were prepared as described above except that cells were plated on poly-L-lysine-coated glass-bottomed plates (Plastek Cultureware,Ashland, MA).

3-cm glass-bottomed dishes were purchased
from MatTek. The glass bottoms were coated with 100 l of FBS
(for untransfected cell lines) or a solution of 0.1% (w/v) gelatin in water
(for 293T cells). 100 l of untransfected cell suspension at 1  105/ml or

We investigated whether mfFLIM could be used to disentangle
the cellular distributions of co-expressed green ¯uorescent
proteins (GFPs) with differing lifetimes (Pepperkok et al.,
1999). HeLa cells were plated on Matek Petri dishes in MEM
supplemented

PHM1-41 cells were plated at 1 x 105 cells/ml on polylysine-coated glass inserts in 35-mm dishes (MatTek, Ashland, MA).

CHO cells (1×105 cells) were spread onto poly-D-lysine-coated glass bottom culture dishes(MatTek Corp., Ashland, MA) and were transfected with 2.5 µg of plasmid by lipofection.

DF-1 cells were infected with the RCASBP M2C
(4070A) virus generated by transfection, as described for the Southern blot
experiments. After passage 1 or at 2 days postinfection, 3  105 cells were seeded
onto 35-mm glass-bottom culture dishes (Mat-Tek

Inner ear
sensory epithelium cultures were prepared from OC, saccule,
utricle, and ampule of P3–P5 C57BL6 mice and from OC of P3
rats. The OC spiral was dissected away from the modiolus in
Leibowitz cell culture medium (Invitrogen). The apical and
middle

Cultured adult myocytes were prepared using common sterile
techniques (8, 25). Isolated myocytes were suspended in serum-free
medium 199 that was supplemented with (in mmol/l) 25 HEPES, 5
creatine, 2 L-carnitine, 5 taurine, and 104 insulin; also inclu

All commercially available chemical reagents
were used without further purification. 9-Fluorenylmethoxycarbonyl
(Fmoc) amino acids and 2-chlorotrityl chloride resin were purchased from Novabiochem (San
Diego, CA). Arginine and lysine were protected by

"GFP–NFAT expressing BHK cells on poly-D-lysine-coated glass-bottomed dish (MatTek, Ashland, MA) were loaded at room temperature with 5 µM Fura-PE3AM (TefLabs, Austin, TX) in physiological salt solution (PSS) containing 150 mM NaCl, 4 mM KCl, 2 mM CaCl2,

The cells were cultured on the coverslips within the glass bottom
of a small cultured dish (No. 0, uncoated, and irradiated. MatTek
Co., USA). At the exponential growth period, the cells were stained
with 10µmol·L-1 fluo-3/AM (Molecular Probe) for 30mi

Cultures enriched in cerebellar granule cells were
prepared using a standard trypsin dispersion protocol (Trenkner 1991; Qiu
et al., 1995). Cerebella from 7-day-old mice (C57BL/6; B&K Universal,
Fremont, CA) were incubated in a Ca2-Mg2-free PBS contai

Bovine capillary endothelial cells were cultured in 10% CO2 at 37 C on gelatin-coated tissue culture dishes in Dulbecco
s modified Eagle
s medium (DMEM, Gibco-BRL) supplemented with 10% calf serum (CS; Hyclone), 10 mM Hepes (JRH-Biosciences), 2 mML

To assess specific labeling of SA-conjugated nanocrystals,
we use HEK293 cells that stably express the cloned
hNET transporter (Galli et al., 1995). The cell culture is
performed as described in Section 2.2.1. To assess the specific
labeling of the An

HK cells (1.5  105 cells per dish) were seeded 1 day before transfection on
glass-bottom culture dishes (35 mm, MatTek, Ashland, MA) to obtain 70%
to 80% confluence. Cells were transfected with CD44-GFP construct using
Effectene Transfection Reagent (

HeLa and 293T cells were grown in Dulbecco's modi®ed Eagle's
medium (Gibco Laboratories, Paisley, UK) supplemented with 10% fetal
calf serum (Helena Bioscience, Newcastle, UK) and 2 mM glutamine at
37°C in a humidi®ed atmosphere containing 5% CO2. Huma

For confocal microscopy, cells were replated at 24 h after transfection
onto 35-mm dishes containing a 1-cm glass-bottomed center well (Plastek Cultureware, Ashland, MA) in growth medium supplemented with 20 mM HEPES buffer, pH 7.4.

CHO-K1 (Chinese hamster ovary) cells were used as the
in vitro target tissue for laser photodisruption. Cells were
cultured in a self-adherent monolayer onto a No. 0 coverslip
in a glass-bottom culture dish (MatTek Corporation,
Ashland, MA). Cells were gr

Isolated intestines were transferred by pipette to a 35-mm Petri dish with a 14-
mm microwell (MatTek Corp.).

"Cells grown on glass-bottomed (35-mm) dishes (MatTek Corp., Ashland, MA) were stained with Hoechst 33342 fluorescent dye (Molecular Probes, Eugene, OR). … Glass-bottomed (35-mm) dishes (MatTek Corp.) with monolayers were prepared for staining with DAF-FM

The procedure for preparation of fluorescently-stained living
cells for microscopic observation was described previously
[34,35]. Briefly, HeLa cells were plated on a 35-mm
glass-bottom culture dish (MatTek Corp., Ashland, MA)
and cultured for 1 day in a

Cells were sub-cultured weekly by trypsinizing and reseeding at 104/cm2. For imaging experiments, fibroblasts were seeded at 500/cm2 on MatTek glass bottom Petri dishes (Ashland, MA, USA) 5–6 days before the experiments.

New Zealand white rabbits (ca. 2 kg) were used for this
study. Cardiomyocytes and cardiac fibroblasts were isolated from adult rabbits by a retrograde collagenase perfusion as
previously described [11]. In brief, rabbits were anaesthetized
by intra-art

Sterile glass-bottom, poly-D-lysine-coated dishes (MatTek Corp., Ashland, MA) were used for translocation assays in COS7 cells.

Glass coverslips (0.12–0.17 mm thick; Matsunami) were treated
with the mixed solution of sulfuric acid and hydrogen peroxide (7:3, v/v) for 1 h at 100°C, washed with
distilled water, and then dried with nitrogen. Dry benzene solution of NPE-TCSP contain

Cells were grown (except for immunoblotting) on glass bottom microwells (MatTek Corporation, Ashwood, MA, USA) coated with collagen type I (Boehringer Mannheim, Brussels, Belgium) and used for experiments upon confluency.

After dissection, the developing sensory epithelium and associated mesenchymal cells were established in an individual MatTek dish (MatTek Corporation, Ashland, MA) as an intact explant culture. For some cultures, the apical 20-25% of the developing epith

The architecture of GFP- and DsRedlabeled
L. pneumophila biofilms was analyzed in glass-bottom microwell dishes
(35-mm petri dishes; MatTek) under static conditions (no medium replacement)
or quasi-static conditions (medium replaced twice a day). An in

For transient transfections on cover-glasses, cells were grown to 90% confluency on sterile glass-bottomed Petri dishes (MatTek, MA, U.S.A.) and incubated with 1 mg of plasmid DNA and LIPOFECTAMINETM 2000 in OPTIMEM (Invitrogen).

For all microscopy,cells were plated, transfected, and imaged in the same 35-mm culture dish that incorporated a no. 1.5 glass coverslip-sealed 15-mm cut-out on the bottom (MatTek, Ashland, MA).

Undifferentiated SH-SY5Y cells (American Type Culture Collection,
Manassas, VA) at passage numbers between 93 and 100
were plated in 35-mm glass-bottom dishes (MatTek Corp., Ashland,
MA) at densities of between 2 and 4 3 104 cells/dish in high-glucose
DME

Primary neuronal culture was prepared
from the cerebral cortex of fetal mice as described previously
(Ma et al., 1991; Tatsumi et al., 1998a). Briefly, the cerebral cortex
was dissected from 15-day-old fetal mouse brain, and the meninges
were carefull

Briefly, dissociated mixed neocortical cell suspensions were prepared...on previously established astrocytic monolayers in either 24-well plates or glass-bottomed dishes (Plastek Cultureware, Ashland,MA).

The murine fibroblast cell line Ltk-, which is highly sensitive to TNF-a, was maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 0.1 mM nonessential amino acids, 5 mM
L-glutamine, and antibiotics (streptomycin,

We examined most cultures without fixation with a confocal microscope [Leica TCS-SP (UV)] or a Zeiss Axiphot fluorescence microscope. For observations of living cells, they were grown in Glass Bottom Microwell Dishes (MatTek; Ashland,MA). However, so

coverslips, which were photo-etched with an alphanumeric grid (Bellco Glass Inc.,
Vineland, NJ) and preglued to 35 mm tissue culture dishes (MatTek
Corp., Ashland, MA).

Normal intestinal or tumor cells were isolated as
described by Evans et al. (43). These cells were then loaded with fura-2
acetoxymethyl ester (10 M) in HEPES balanced salt solution containing the
following components (in mM): 138 NaCl, 1.8 CaCl2, 0.8

HEK 293 cells and cultured myocytes expressing fluorescent
PKCs were plated onto glass-bottom culture dishes from MatTek Corp. (Ashland, MA) and cultured for 30–40 h before
observation. Before imaging cells were washed twice with
serum-free culture med

Cerebellar cells were purified by using a modified procedure described by Hatten (Hatten, 1985). Cerebella were dissected from P6 animals. After removal of meninges, cerebellar tissue was treated with Trypsin-EDTA (Gibco-BRL) and triturated into single

WIF-B9 cells
were cultured in a humidified 7% CO2 incubator at 37°C (14).
Unless otherwise specified, cells were plated on a 35-mm glass
bottom dish (MatTek) at 1.4  105 cells per dish in complete
medium. Infections with recombinant adenoviruses enco

An asymmetric Jurkat cell was trapped using the HOT and scanned back and forth across the
Raman excitation beam in a 5×5 step scan (stepsize 3 µm) and a spectrum was taken at each point. To show the advantage of using optical tweezers for Raman spectrosc

Cultures were plated on glass-bottomed dishes (Mattek Cultureware, Ashland, MA) and mounted to a stage adapter on an
inverted microscope (Nikon Diaphot, Tokyo, Japan).

For direct visualization under an inverted
fluorescence microscope (Leica DMIRB), Vero–ICP10-EGFP cells were seeded on a bottom glass microwell dish (MatTek Corp., Ashland, MA) aday before infection. Cells were infected with HSV in DMEM without phenol r

For imaging, suspensions of GT1-1
or GT1-1 trk cells were diluted and plated onto 35-mm culture
dishes (Mat-Tek, Ashland, MA) possessing an oval cut-out
sealed by a glass coverslip coated with poly-D-lysine: laminin
(12). Cultures were analyzed when ,

Cultured adult myocytes were prepared using a sterile technique described previously (7).
Isolated myocytes were suspended in serum-free Medium-199 which was supplemented with
(mmol/L): 25 HEPES, 5 creatine, 2 L-carnitine, 5 taurine, and 10-4 insulin. 0

A431 and COS-7 cells were cultured in DME supplemented with 10%
FBS. The transfection of GFP or RFP fusion protein constructs were
made using cells in a 60-mm dish (Nunc), a glass-bottomed 50-mm dish
(MatTek Corporation), or on 22-mm-diameter coverslip

Imaging experiments were performed as previously described
(Takasuka et al. 1998, McFerran et al. 2001, Norris
et al. 2003). Cells were seeded (5104 and 1105
cells/dish for GH3 and primary PD cells respectively) onto
35 mm coverslip dishes (MatTek C

aT3 cells stably expressing wild-type
a1bAR and a1bAR/GFP were seeded at 1 3 105 per well of the
cover glass-bottom culture dish (MatTek Corp., Ashland, MA)
in 2.0 ml of medium and examined using LSM-GB200 within
30 min at room temperature.

For microinjection, cells were seeded in 35-mm tissue culture dishes with a glass-bottomed microwell (P35G-1.5-14-C, No. 1.5, uncoated, MatTek Corporation, Ashland, MA) at 30–40% confluence 24 h prior to microinjection.

P13.9 cells were plated in sterile glass-bottom 35-mm dishes coated with
poly(D-lysine) (MatTek, Ashland, MA) at a density of 1  107 cells/dish.
After overnight adherence, the cells were infected with VV-CD1d1WT,
VV-CD1d1TD, or VV-CD1d1Y322A at an MOI

Coverslip-grown MDCKT cells were infected with ts045 VSV-G-YFP and induced
with 5 mM butyrate for 16 h at 40C. Tfn uptake was performed as
described above. Cells were incubated 2 h in media at 20C (last hour in
media plus 0.1 mg/ml CHX). Coverslips w

For a given experiment, RBL cells were plated at a density of 1.15 x 10^5 cells/mL on glass coverslip-bottomed grid dishes (P35G-7-c-grid; MatTek, Ashland, MA) and allowed to adhere overnight. Adherent cells were exposed to intense near-infrared illuminat

On day 0, cells were set up in glass-bottomed 35 mm dishes (MatTek Co., Ashland, MA) at a density of 3 3 104 cells/dish in medium A supplemented with 5% FCS. On day 2, cells were washed with PBS and refed 1 ml of medium A supplemented with 5% newborn calf

MDCK cells were grown on glass-bottomed 35 mm tissue culture dishes (MatTek) (1×104 cells/cm2), transfected with the fusion constructs and incubated in serum-free DMEM overnight to quiesce the cells.

· 103 cells/well were seeded into MatTek Cultureware (MatTek Corporation,
USA). After a 4 h incubation at 37 C, 1 ml fresh medium
was supplemented. At 24 h post-transfection, cells were serum-deprived
for 20 h and stimulated with 10 ng/ml EGF for the

For determination
of pHi, cells were plated on 35-mm microwell plastic
dishes with glass coverslips glued to 1-cm diameter holes in
the center of each dish (Mattek Corp., Ashland, MA). For
melanoma cell experiments, coverslips were coated with a
mixt

Ovaries were collected from 20–25-d-old mice. Preantral follicles and early antral/antral follicles were dissected from ovaries. 4–10 follicles were placed on Millicell culture plate inserts inside coverslip-bottomed dishes containing 1.6 ml of medium. Th

For confocal microscopy, 2-4 x 104 COS-1 cells were plated in 35 mm glass-bottom dishes (MatTek Corp., Ashland, MA) and cultured overnight at 37 °C in DMEM supplemented with heat-inactivated FBS, and 50 µg/ml Pipracil at 37 °C under 5% CO2.

G-actin solutions were prepared by
dissolving lyophilized G-actin in deionized water and dialyzing
the solutions against fresh G-buffer (2 mM Tris
HCl
0.2 mM
ATP
0.2mMCaCl2
0.2mMDTT
0.005% NaN3, pH8.0) at 4°C
for 24 hr; the buffer was replaced with fr

Infected
or treated cells [where infections or treatments were
performed on glass coverslips in MaTek (Ashland, MA)-brand
dishes] were washed with PBS to remove trace serum
medium.
Monolayers were fixed with 3.7% formaldehyde in 1
Fix Buffer
pH 7.2 (

E14 dorsal retinal explants were grown overnight in 35 mm
coverslip culture dishes (MatTek Corporation). Cultures were
overlaid with prewarmed mineral oil (Sigma or Fisher) and
equilibrated on a 37°C microscope stage incubator with CO2 influx.
Glass m

Cerebella of
postnatal day 0–3 (P0 –P3) mice (CD-1) were placed in ice-chilled HBSS
and freed from meninges and choroid plexus (Komuro and Rakic, 1996,
1999; Yacubova and Komuro, 2002a; Kumada and Komuro, 2004). Cerebellar
slices were then made with a

Rat superior cervical ganglia were dissected from postnatal
days 0–2 pups with the use of #5 watchmakers forceps and a Zeiss
(Oberkochen, Germany) SV6 stereomicroscope, collected in chilled L-15
medium (Invitrogen, Carlsbad, CA), and then treated for 1

The cells were cultured for 24 h on 35-mm glass-bottomed dishes (MatTek, Ashland, MA) in minimal essential medium containing 10% fetal calf serum in 5% CO2 in air at 37°C.

Preadipocytes were inoculated in DMEM/Ham’s F-10 medium (DMEM-F10) (1:1, vol/vol) containing 10% FBS, 15 mM HEPES, and antibiotics at a density of 30,000 cells/cm2. Confluent monolayers of preadipocytes were induced to differentiate with a standard differ

Cells were plated either onto glass bottom dishes (MatTek, Ashland, MA) or on autoclaved coverslips (Fisher Scientific, Fair Lawn, NJ).

Transfected 293T cells were grown
on 35-mm-diameter coverglass-bottom dishes (MatTek Corporation, Ashland,Mass.) and processed at room temperature for immunofluorescence staining 48 h
after transfection.

EC were collected from the basilar artery of male Sprague-Dawley
rats aged 4 to 6 weeks. After the rats were anesthetized with diethyl
ether, they were decapitated, and the basilar artery was quickly
removed under sterile conditions. The arterial segme

The SMG were cultured on membranes at the air/medium interface at 37°C for 1–72 h. Five or more glands were placed on Nuclepore Track-Etch Membranes (Whatman, Clifton, NJ) (13 mm, 0.1 µM) floating on 250 µL of DMFTV standard media [DMEM/F12 (1:1) (Invitro