Problems stimulating CD4s with plate bound anti CD3 - (Aug/28/2010 )
Hi all,
I知 putting together a suppression assay for Tregs and I知 stumbling on the stimulation step.
I知 using either fresh or frozen PBMCs simply to stimulate CD4s with plate bound anti CD3 (OKT3 NA/LE from BD).
Here痴 what I do:
Flat bottom 96 well plate coated with 50ｵl anti CD3 (0.5ｵg/ml) in PBS for 2h at 37ｰC.
Wash 3 times with PBS.
Add about 105 PBMCs labeled with 1ｵM CFSE in complete RPMI (10% FBS, PenStrep, Sodium Pyruvate, 2ME)
Incubate for 3 days at 37ｰC 5% CO2.
Stain with anti CD4 and run on flow cytometer.
Since I知 having problems I titrated the anti CD3. Turns out it works but I need to jack up the anti CD3 to at least 10 ｵg/ml to see anything but I only get good proliferation (as in more than 50% of the CD4s) for 50 ｵg/ml. That痴 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知 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知 having problems I titrated the anti CD3. Turns out it works but I need to jack up the anti CD3 to at least 10 ｵg/ml to see anything but I only get good proliferation (as in more than 50% of the CD4s) for 50 ｵg/ml. That痴 basically 10 to 100 times what I usually see in protocols.
5-10ｵg/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.