I am isolating lymphocytes from lymph nodes of C56BL/6 mice. After the isolation I activate them with 4ug/ml of soluble anti-CD3 (clone 2C11) and 1ug of soluble anti-CD28 (clone 37.51) and 24h later I check the viability by flow cytometry using Propidium Iodide. However, I am observing some variability in the % of PI negative cells, ranging from 35 to 80%. Is this normal? How to achieve a high viability (70-80%)? Thanks in advance.
Hi,
You should expect some cell death, but 35-80% is pretty high. You can try scaling back the amount of CD3 to 1ug/ml. Right now you might be causing activation induced cells death. The cells will still be activated just fine with 1ug/ml CD3.
you should take care of removing as much as much fat as possible from your LNs before cell suspension and use 2C11 coated on plastic to activate your cells (don't need CD28 in that case but little IL-2 can help). Also use Live/dead coloration to monitor cell death, PI give lousy stainings and is toxic to the environment. Cheers.
I always use anti-CD3 coated on plastic plus anti-CD28 in the medium and get good results with CD4+ T cells. Did you check the viability right after your isolation?
Thanks for all the replies! But here is the problem: after the activation I am electorporating these cells with Nucleofector. I found that activated T cells work better, and I made a titration of the anti-CD3. Using 4ug/ml the viability post-nucleofection almost doubled. I don´t think it is causing AICD, because cells expand normally after the nucleofection.
@Doris: Initially I tried anti-CD3 coated on plastic. I´ve made a 5ug/ml solution and coated overnight at 4ºC. However the cells were not as much activated as using soluble anti-CD3, but I don´t know why...I tried to coat directly the 2C11 to the plastic, but some people coat first with anti-IgG and after with 2C11, maybe was this...
@Gilles: What live/dead colorotaion do you recommend?? Indeed the PI staning isn´t very good...
@Fabien: I am using total lymphocytes from lymph nodes.
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