Planning+ELISA+Experiment+2-6-12

Planning ELISA Experiment 2-6-12

Do I have Ova peptide and enough of it? How does Shen usually do the experiment? Timing of Experiment Layout of Experiment > 3 splenocytes + target cells > 3 splenocytes only > 3 target cells only Protocols Followed
 * I have 100 uL of 1 mg/mL peptide. How much do I use typically use to pulse each sample?
 * see Killing Assay. I typically use 10 ug/mL peptide. Also, I typically use a 10:1 effector to target ratio (10^6 effectors and 10^5 targets) in a 200 uL volume.
 * How much peptide would I add to each well? (1000*ug/mL*y/(200*uL)=10*ug/mL -> y = 10*200/1000 = 2 uL. It looks like I have plenty of peptide
 * 20 ug/mL peptide used
 * 200 uL volume per well
 * Shen makes 1*10^7 cell/mL stock, a 40 ug/mL peptide stock, and then he mixes 100 uL of each together for his final well
 * Setup Wednesday night
 * start 8:30am Thursday (finish sometime Thursday afternoon)
 * 3 splenocytes + target cells + peptide
 * Target Cells: CRL 2116 Mouse Balb/c breast cancer cells
 * Peptide: Ova peptide
 * Start pulsing target cells with peptide (detach cells with trypsin, count them, put the appropriate amount into wells, add peptide to some of them them (3 of them))
 * Surgical Removal of Spleens
 * Splenocyte Preparation Protocol
 * Combine target cells, peptide, and splenocytes into wells appropriately
 * Perform ELISPOT with Shen, I don't have the protocol for that yet.