T+cell+assay+with+no+target+cells+in+5+day+induction+and+no+purification+of+T+cells+with+Robosep+12-7-11

Spleens harvested on 12-6-11 around 5 pm. Note that it would have been better for us to have added the peptide at this time, but we did not do this. The splenocytes were suspended in complete DMEM and split in 1/2 into two T-25 flasks with 5 mL in each.

12-7-11 1551 We added 3 mL more complete DMEM.

We added 80 uL of peptide stock.
 * 1000*ug/mL*y/(8*mL) = 10 ug/mL -> y = 0.08 mL = 80 uL

We made a 1/10 dilution of IL-2 in complete DMEM and added 1 uL.
 * 4*10^6 U/mL*y/(8 mL) = 50 U/mL -> y = 0.0001 mL = 0.1 uL

12-9-11 Removed some media and added more IL-2

12-13-11 Performed killing assay using BALB/c 3T3 fibroblast cells and unpurified splenocytes. 3 Samples were used 1 targets with peptide 2 targets without peptide 3 no effectors and no peptide

1*10^6 effectors were mixed with 1*10^5 targets in sensitization DMEM media in 1.5 mL in 5 mL flow cytometry tubes for 4 hours. Cells were then stained with caspase.

12-13-11

More IL-2 (50 U/mL) and peptide (10 ug/mL) was added to remaining splenocytes that were not used in the killing assay.

__Pictures of T cells after 7 day Incubation__ S:\Research\CTL\Experiments\Splenocytes after 7 day incubation 12-13-11

__Flow Cytometry Results__ S:\Research\CTL\Experiments\Killing Assay Flow Cytometry Results 12-14-11

Note: I had a sheath pressure error. I called the service number and they told me to tighten the caps for the sheath and cleansing fluid tanks more tightly which solved the problem. I spoke with Laurine who gave me the following number 357836.