Version+of+file+for+indexing+8-22-12

Summary of Ova CTL Experiment 8-21-12 A CTL chromium release assay with B16F10 target cells pulsed with SIINFEKL peptide from the Ova antigen and effector cells from Ova immunized mice did demonstrate that the target cells were killed in a dose dependent manner with specific p values compared to controls. However, the current CTL chromium release assay protocol needs to be optimized. This is actually the 2nd time I performed the chromium release assay. The first time, I made a pipetting error which affected many samples, and I also observed some results I could not fully explain. I suspected but could not prove that DMSO alone was killing my target cells. In this experiment, I had all of the controls necessary to prove that this was the case. There are still some things I wasn’t fully expecting from this experiment though. I did not expect that effector cells would kill non-peptide pulsed tumor cells in a dose dependent manner. Now that I see the result this makes sense to me though since effector cells probably have some general ability to recognize some features of tumor cells. I also did not expect the negative control peptide sample to exhibit greater killing at low E:T ratios and less killing at higher E:T ratios. Note that in this protocol my target cells were incubated with the chromium for about 5 hr rather than the 1-2 hr that the protocol suggests. This happened because I thought I had enough effector cells from the previous experiment, but this turned out not to be the case. Therefore, I obtained some more splenocytes from another Ova immunized mouse which took time. I have two sets of data one for a 6 hr incubation plate and one for an overnight plate. Note that all p values were calculated by comparing a certain E:T sample with the corresponding target with peptide dissolved in water and DMSO or DMSO only. E:T Ratios E:T Ratio Target Cell Number Effector Cell Number 100 1.00E+04 1.00E+06 30 1.00E+04 3.00E+05 10 1.00E+04 1.00E+05 3 1.00E+04 3.00E+04 Sample Name Key TONPND = Target cells only not pulsed no DMSO TONP= Target cells only not pulsed TOP = Target cells only pulsed TONCP = Target only negative control pulsed (negative control = CPV172 peptide) EO = Effector Cells only EOP = Effector cells only with peptide EONCP = Effector cells only with negative control peptide TNPEX = Targets non-pulsed with effectors at E:T ratio X:1 TNCPEX = Targets negative control pulsed with effectors at E:T ratio X:1 TPEX = Targets pulsed with effectors at E:T ratio 100:1 6 hr Incubation Results Overnight Incubation Results Ways to Optimize Protocol · Use as little DMSO as possible or even no DMSO if the peptide can be soluble without it. Alternatively, try incubating the target cells with peptide in a larger volume (rather than approximately 100 uL). · Resuspend all pelleted target cell samples in a specific volume. The protocol I followed said to take away the supernatant and leave about 100 uL left. However, I think I ended up with varying volumes for my 3 types of target cells (non-pulsed, ova peptide pulsed, and NC peptide pulsed). I think it would have been better to take away just about all of the liquid and resuspend all target cells in a known volume (probably 1-5 mL RPMI rather than 100 uL). The media was starting to change colors after the incubations, and I think the cells may not have had enough nutrients in the small 100 uL volume of media. · Don’t let the target cells incubate with chromium for longer than 1-2 hr. · When transferring 100 uL of supernatant to the Luma plate for counting, make sure the tips are firmly attached and a full 100 uL is transferred to the Luma plate for all of the tips on the multichannel.