Isolation of CD4+CD45RA+ T cells with high purity is essential for immune functional assays. Can you please share your experience with isolation of CD4+CD45RA+ (using Miltenyi isolation kits), regarding the maximum number of cells from one buffy coat.
I would appreciate if you can share your information in cases where actual purity analysis was made after isolation by flow cytometry.
Thanks
A buffy coat can be derived from a 10 ml vacutainer of blood to a 20L apharesis. The T cell content then depends on the age of the donor, fewer lymphs with age and also the condition of the donor. Cancer, ID or autoimmune patients will have fewer lymphs. Perhaps a better question is what is the yield following a Miltenyi isolation of CD45RA+CD4+ cells.
Hello,
Human naive CD4+ T Cells typically represent between 1-10% of leukocytes in healthy normal blood samples. As mentioned above, there are a number of factors that may alter this frequency as well as the cell isolation efficiency.
If you want better purity, please do FACS sorting of CD4+CD45RA cell which will give purity even above 98%. all the best
I have used Miltenyi naive T cell isolation kit, with >97% purity in most experiments. The contaminating cells seemed to be mostly CD4-CD8- CD3+ T cells. More than 99% of CD4+ T cells were CD45RA+RO-.
I used the LD columns to get this increased purity, rather than the LS which are recommended by Miltenyi.
I would usually use up to 100*10E6 cells per column and a usual yield would be 2-6 million, so about 2-6%.
Please see following paper for example purity we obtained:
http://www.ncbi.nlm.nih.gov/pubmed/24122358
Jolien, Good answer. I note that you used a buffy coat which can cover a host of sins. I used to get buffy coats from the red cross that were from a unit of blood and considerably more than 100e6. However, you reported the starting number so its all good.
It might be interesting to provide a yield based on starting number of naive T cells (or an average).
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