Hi
I did isolation of PBMCs from whole blood through ficoll and activated them using anti-cd3/cd28 antibodies,
after several days I did staining using flow cytomtery with cd4 and cd8 fluorochromes.
When I got the results, I noticed populations of double positive and double negative (CD4/CD8)
Any explanation for those groups.?
Thanks
Do you isolate T-cells before activation or use whole PBMCs? Without T cell isolation my guess will be nonspecific antibodies binding to monocytes or insufficient wash after flow cytometry staining. Do you use any kind of block before or within the staining (Fc block, or antibodie's host serum)?
Hi Alpha, most likely your findings are totally correct. Human (and mouse) blood contains a small but clearly detectable CD3+ CD4-CD8- double negative T cell subset which is known as gamma-delta T cells (opposed to the alpha-beta T cells which are either CD4 or CD8 positive). The designation gamma-delta (gd) refers to a distinctive T-cell receptor. The cells have an important function as 'first line defense' in infections.
Likewise, some individuals also have a sizable number of CD4+CD8+ double positive T cells. I attached a paper for you on the role of this T cell subset in health and disease. I hope this helps!
Hi Natallia Mikhalkevich, and thanks for your comment.
Actually I did isolation of T cells using the ficoll method, but DIDNOT not block the Fc.! do you think other cells like B-cells might be a reason for this ?
Thanks
Hello Christian Hundhausen, and thanks for your information and I will go through that paper
Thanks again for your help
Hi Alpha, Ficoll gradient isolation will not provide a pure population of Tcells. Ficoll collects lymphs and monos in the interface. Unless you specifically isolated Tcells post-ficoll, your sample will contain monos, Bcells and NK cells in addition to your desired Tcells. Try Miltenyi column isolation with CD3 to obtain a pure Tcell population.
Once you have your purified Tcell population, you will continue to see the 3+4+8+ and 3+4-8- cells as described by Christian.
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