Technical Reference #1898

1898.

Heat Shock Protein 70B' (HSP70B') Expression and Release in Response to Human Oxidized Low Density Lipoprotein Immune Complexes in Macrophages Kent J. Smith; Waleed O. Twal; Farzan Soodavar; Gabriel Virella; Maria F. Lopes-Virella; and Samar M. Hammad, Medical University of South Carolina, Journal of Biological Chemistry, 285(1898), (2010)
Link To Paper

Materials & Methods:
Expression of GFP-tagged human HSP70 and HSP70B — HSP70 and HSP70B cDNAs were purchased from OriGene (Rockville MD) and used to generate a 1700-bp PCR amplicon using high fidelity Taq polymerase (Roche Applied Science) and custom designed primers (Table 1) with KpnI and ApaI restriction enzyme sites. The PCR products were cloned into a shuttle vector using the TOPO TA cloning kit (Invitrogen) and transformed in OneShot Top10 competent cells (Invitrogen). The product was sequenced in both directions to verify the clone. The cloned HSP sequences were then cloned into the pEGFP-N1 expression vector (Clontech Mountain View CA) coding for an enhanced green fluorescent protein. Ligation of HSP70 or HSP70B sequences was confirmed byDNAsequencing. Transfection with LipoD293TM DNA in vitro transfection reagent (SignaGen Laboratories) was performed according to the manufacturer’s instructions. Briefly 1 g of plasmid DNA and 3 l of LipoD293TM reagent were each diluted into 50 l of serum-free Dulbecco’s modified Eagle’s medium. The mixtures were then combined and incubated for 15 min at room temperature. RAW 264.7 cells (80–90% confluent) were incubated with the combined mixture under normal growing conditions for 18 h. Transfection reagent-containing medium was then replaced with complete medium and incubated for an additional 24 h prior to treatment. The amount of green fluorescence was quantified as described above. For live imaging cells were maintained in serum-free treatment-containing medium in glass bottom 96-well plates (MatTek Corp. Ashland MA) at 37 °C and 5% CO2 for the duration of the experiment.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
human monocytic cell line U937