Planning ELISA Experiment 2-6-12

2015-01-13

azim58 - Planning ELISA Experiment 2-6-12

Do I have Ova peptide and enough of it?

* I have 100 uL of 1 mg/mL peptide. How much do I use typically use to
  pulse each sample?
  o 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.
  o 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

How does Shen usually do the experiment?

* 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
Timing of Experiment

* Setup Wednesday night
* start 8:30am Thursday (finish sometime Thursday afternoon)
Layout of Experiment

* 3 splenocytes + target cells + peptide
  3 splenocytes + target cells
  3 splenocytes only
  3 target cells only
* Target Cells: CRL 2116 Mouse Balb/c breast cancer cells
* Peptide: Ova peptide
Protocols Followed

* 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.

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