Dear Alex, We stain for intracellular TGFb ligand using a rabbit polyclonal antibody from Santa Cruz ((Ab-V) #sc-146), as well as the surface receptors (RI, T-19, #sc-402 and RII, C-16, #sc-220), again using Rb polyclonal antibodies. These work well on cultured cell lines as well as paraffin embedded tissue. We use fixed cells so we are not staining viable cells for TGFb synthesis in the way that you may measure other cytokines (IFN etc). For flow cytometry we fix cells (EDTA harvested) for 5 mins at RT in 1% paraformaldehyde in PBS; wash; fix 70% methanol overnight. Both reagents and the cells are stored at 4oC. Using 0.5x10^6 cells per test we wash twice in PBS, then once in PBS+1%serum. Then stain with 10ul Ab for 1hr, wash (PBS+1%serum), and label using 100ul 1:100diluted Sigma FITC goat anti- rabbit IgG (whole molecule) #F-0382 for one hr, wash then we counterstain with propidium iodide (300ul 50ul/ml in PBS) plus RNase (50ul 1ul.ml PBS). However, you could just resuspend cells in PBS. Hope this helps. Best wishes, John
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