I want to activate CD4+ T cells (from OTII mice) in the presence of IL-27 using OVA peptide. I would like to use a suspension of whole splenocytes to do this instead of BMDCs cultured with OVA specific CD4 T cells. Has anyone successfully managed to use whole splenocytes? If so, would you share the details with me please?
So far, I have used 1 million whole splenocytes with concentrations of OVA peptide from 1 - 10ug/ml but I don't see any activation after three days of culture. Just dead cells :( Any feedback would be very much appreciated. Thanks!
I have done this several times using whole splenocytes from OT-II mice. For this spleens were obtained from OT-II mice and single cell suspensions were generated by pressing spleens through a nylon mesh using the plunger of a syringe. RBCs were lysed using 0.8% ammonium chloride (RBC lysis buffer. Cells were cultured in U-bottom 96 well culture plates at a concentration of 2-4x10^5 cells/well. The culture conditions used were RPMI supplemented with 10% FBS, sodium pyruvate, glutamax, 2-ME, and Gentamicin. Peptide, OVA(323-339), was added at concentrations of 1 ug/ml or 10 ug/ml for 48 and 72h at 37C, 5% CO2. I measured proliferation by 3[H]-thymidine incorporation, as well as, collecting supernatants for analysis of cytokine production by ELISA. I didn't look for surface marker expression. It worked quite well under these conditions. I don't know what the influence of IL-27 would be on the culture, but perhaps it could be leading to AICD. I assume however, that your seeing the same issue in control cultures that lack IL-27. One thing you should try is throwing in whole OVA (100ug/ml) with the cells to make sure that the issue is not with your peptide. If the cells react to whole OVA then something is wrong with your peptide. If they don't react to OVA either, then you may want to re-genotype your mice, or inject them directly with OVA/peptide and look for in vivo reactivity to see if your having issues there.
OTII TCR is specific for chicken ovalbumin peptide 323-339. Does your peptide have this region?
Presentation of the peptide by MHCII is required for the signal transduction by CD4, without this, the cellular activation would be impaired.
I have done this several times using whole splenocytes from OT-II mice. For this spleens were obtained from OT-II mice and single cell suspensions were generated by pressing spleens through a nylon mesh using the plunger of a syringe. RBCs were lysed using 0.8% ammonium chloride (RBC lysis buffer. Cells were cultured in U-bottom 96 well culture plates at a concentration of 2-4x10^5 cells/well. The culture conditions used were RPMI supplemented with 10% FBS, sodium pyruvate, glutamax, 2-ME, and Gentamicin. Peptide, OVA(323-339), was added at concentrations of 1 ug/ml or 10 ug/ml for 48 and 72h at 37C, 5% CO2. I measured proliferation by 3[H]-thymidine incorporation, as well as, collecting supernatants for analysis of cytokine production by ELISA. I didn't look for surface marker expression. It worked quite well under these conditions. I don't know what the influence of IL-27 would be on the culture, but perhaps it could be leading to AICD. I assume however, that your seeing the same issue in control cultures that lack IL-27. One thing you should try is throwing in whole OVA (100ug/ml) with the cells to make sure that the issue is not with your peptide. If the cells react to whole OVA then something is wrong with your peptide. If they don't react to OVA either, then you may want to re-genotype your mice, or inject them directly with OVA/peptide and look for in vivo reactivity to see if your having issues there.
