Problems stimulating CD4s with plate bound anti CD3 - (Aug/28/2010 )
Hi all,
I’m putting together a suppression assay for Tregs and I’m stumbling on the stimulation step.
I’m using either fresh or frozen PBMCs simply to stimulate CD4s with plate bound anti CD3 (OKT3 NA/LE from BD).
Here’s what I do:
Flat bottom 96 well plate coated with 50µl anti CD3 (0.5µg/ml) in PBS for 2h at 37°C.
Wash 3 times with PBS.
Add about 105 PBMCs labeled with 1µM CFSE in complete RPMI (10% FBS, PenStrep, Sodium Pyruvate, 2ME)
Incubate for 3 days at 37°C 5% CO2.
Stain with anti CD4 and run on flow cytometer.
Since I’m having problems I titrated the anti CD3. Turns out it works but I need to jack up the anti CD3 to at least 10 µg/ml to see anything but I only get good proliferation (as in more than 50% of the CD4s) for 50 µg/ml. That’s basically 10 to 100 times what I usually see in protocols.
Also tried with CD4s sorted using MACS magnetic sorting... same result.
Can anybody tell me what it is I’m doing wrong? What is inhibiting proliferation in my assays? any ideas?
I would appreciate any help, thanks!
Max
mxdsmrts on Sat Aug 28 16:09:57 2010 said:
Since I’m having problems I titrated the anti CD3. Turns out it works but I need to jack up the anti CD3 to at least 10 µg/ml to see anything but I only get good proliferation (as in more than 50% of the CD4s) for 50 µg/ml. That’s basically 10 to 100 times what I usually see in protocols.
5-10µg/ml is a total normal concentration to activate your tcells. A lot of protocols using this concentration. I think you should incubate a bit longer. Usualy I incubate for 5 days.