peptide pulsing for CTL assays - (May/04/2001 )

Hi All, Could anybody tell me how much peptide would one use for pulsing target cells for CTL assays? I would greatly appreciate the protocol for peptide pulsing too. One more question: How efficient is electroporation for transfecting mammalian cells? How messy is it?Thanks in advance.Lakshmi

I usually use 10ul of a 0.2 mg/ml to pulse up to 5 million cells. That's usually sufficient for screening for activity. As for electroporation, it's not messy, but efficiency can vary depending on how hardy your cells are. I've heard that Fugene works well, but haven't tried it myself.

electroporation of P815 works very well atleast with the construts I am using. I have tried once with the PBMC but didn't work at all. The cells all of them died in the process and ruined my expt. I am in the process of standerdising it now and will let the forum know if I hit the right conditions for these.

Devesh.