Killing+Assay+with+different+abs+and+dyes+11-17-11

Splenocytes from Balb Ova immunized mice were mixed with Ova peptide pulsed target cells (Balb 3T3 and Balb CRL 2116).



For the most part, our standard protocols were followed. 11-18-11 We added IL-2 to the mixed cultures one day after starting them. We added 1 uL 100 ug/mL rh-IL-2 to 20 mL culture. 11-20-11 I added IL-2 to the mixed cultures 3 days after starting them. Added 3 uL 100 ug/mL rh-IL-2 to 30 mL culture (10 more mL of DMEM sensitization media was added to the 20 mL culture which looked fairly yellow).

Results from the killing assay from the splenocytes used immediately Results 11-18-11

11-21-11 When we tried to purify our T cells with the Robosep after 4 days of incubation, we only had about 100,000 cells which was not enough. We hypothesize that there could have been several reasons that we lost so many T cells. One reason is that there was a lot of clumping that was hard to get rid of. The Robosep may not have purified the cells appropriately because of this clumping. Another possible reason we did not obtain good results may have to do with the age of the Robosep T cell enrichment kit we used. We found a large number (maybe about 20) old T cell enrichment kits from 2006 (5 years old), and we used some of these kits. Due to the age of these kits they may no longer be effective.

The next time we do an experiment for these CTL assays, we will most likely take the spleen from a normal mouse, use a fresh Robosep kit, and then perform some flow cytometry with CD8, CD3, and CD4 antibodies to make sure that we have purified killer T cells and not helper T cells or any other types of cells.

We also plan to become better at identifying which cells are the T cells in our mixed cultures. We plan to do this by finding out the diameter of a T cell, taking some images of our cultures, and placing a black scale bar on these images so that we can characterize the different types of cells we are looking at.

The next time we plan to do an actual killing assay, we are considering culturing the splenocytes by themselves and just adding IL-2 (instead of culturing the splenocytes with target cells). This way most of the splenocyte cells would be in suspension and may be easier to handle.

__Some details from experiment__
 * Protocols followed
 * Last part of "Inducing cytolytic activity in CTL precursors"
 * Purification of T cells with Robosep
 * Catalog number of old T cell enrichment kits found: 19753A
 * Pellet was resuspended in 3 mL Robosep buffer before starting robosep protocol
 * samples were too clumped to count
 * Counts for targets
 * peptide: 207/4*2*10^4
 * no peptide: 309/4*2*10^4
 * Count of effectors after Robosep purification
 * 25/4*2*10^4 = 125,000