I need to culture retrogenic Ag specific CD8 cell clone with splenocytes in presence of an Ag (Class I dependent peptide) and check the proliferation and IFN-gamma expression by flow cytometry. Can anybody share the protocol for it? What should be the E:T ratio to be used, conc. of CFSE to be used and incubation time of cells with Ag (peptide)? Please suggest
Hi
You have to check the E:T ratio with different ratio of effector and T cells I use to do in 1:2.5 to 1:50 check for the proliferation and IFN gamma secretion in each ratio.
For CFSE I use 3microM for 1-10million cells in PBMCs incubate for 5min at 37C, then make it up with serum containing media and incubate for another 15min at RT after that wash and suspend in appropriate volume.
For IFN gamma you have to check at different hours say from 6 to 48h. IFN gamma will secrete at 6h while using PMA use Golgi stop to prevent the secretion of IFN gamma, you can use saponin and paraformaldehyde to fix and perm the cells.
Hi Hemavathi....Thanks for the info. Since I am doing this type of culture for the first time, can you please give me an elaborate protocol. I am not sure about many things like do I have to stain cells with CFSE before antigen treatment and do I need to treat both the effector and target cells together with CFSE and antigen or treat the target cells with CFSE and antigen alone, before mixing them with effector cells for culture. How many days are appropriate to culture these cells for observing the Ag presentation or check the CFSE proliferation?
Hi
Stain the target cells with CFSE for 5min in PBS and make sure no clumps in your cell preparation, after that make it up with media and rest it for 15 min at RT and wash again with media. After washing rest the cells overnight or 4h in incubator. Then you can use the target cells to coculture with your antigen or antigen presenting cells. I use to culture the cells for 10days because Ag we use is not strong stimulator. So it is depend on stimulating capacity of antigen. While doing use appropriate controls like Unstimulated but stained with CFSE and use strong known stimulant as positive control.
Good luck
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