Technical Publications Live Cell Imaging

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MATERIALS & METHODS

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

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

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.

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

Briefly, cells were cultured in 35-mm glass-bottom dishes (MatTek,
Ashland, MA) for 2 days.

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.

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).

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

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.

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).

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).

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 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.

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 tracheas were then placed in F12 medium and transferred into a 35-mm experimental chamber with a glass
bottom (Plastek Cultureware, Ashland, MA).

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).

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).

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.

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).

Caco-2 cells were seeded at a density of 1 3 105 cells/cm2 onto 35-mmdiameter
glass-bottomed coverslip dishes (MatTeck Corp.) and cultured for 5 to
7 days.

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.

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.

For multi-channel TIRFM, CHO Fc?RII cells grown in 35-mm glass bottom dishes (MatTek, Ashland, MA)…

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).

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.

The pellet was then resuspended in DMEM containing 10% heat inactivated horse serum and 5% fetal bovine serum (Sigma), and plated on…35 mm Petri dishes with 10 mm glass bottom microwells (Mattek Corporation, Ashland, MA) for imaging experiments.

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.

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…

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

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.

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).

Cells used for FRET-imaging were grown on 6 cm culture dishes with a coverslip on the bottom (Mattek Corp.).

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).

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

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).

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.

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

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.

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

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.

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.

Dispersed luteal cells (106 cells/dish) from each of four heifers were
cultured in glass-bottom dishes (MatTek, Ashland, MA) for 24 h before
transfection.

Embryos were then imbedded in a coverslip-bottomed petri dish (MatTek Corporation), in 0.5% low melting point agarose dissolved in embryo medium with 0.02% tricaine.

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)…

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.

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

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).

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

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

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.

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).

After incubation, tissues were washed in Ca2+-deficient solution, and placed in a dish with non-fluorescent glass at the central area (MatTek), then dissociated using fine needles.

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).

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

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.

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).

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.

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

For live cell imaging, HEK cells were grown in 60 mm culture dishes and, 24 hours post-transfection, were passed
at a 1:4 dilution onto collagen coated glass bottom 35 mm dishes
(MatTek, Ashland, MA).

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.

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

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

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

Time-lapse imaging of live embryos was done on an inverted Zeiss LSM5 Pascal with a 20/0.7 n.a. air lens. Live embryos were collected at 29C to maximize UAS-transgene expression, dechorio-nated in 50% bleach, washed in 0.01% TritonX-100 and water, and s

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 ,

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

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.

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

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

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

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