Planning ELISA Experiment 2-6-12

2015-01-13

azim58 - Planning ELISA Experiment 2-6-12

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.