I wish to isolate Tgamma delta 17 cells from human PBMCs. further want to culture them. Does anyone know any feasible method?
Dear Dr. Bernd Zorr, Thank you for your valuable suggestion but Life technology does not have dynabeads for gamma delta T cells and more specifically not for IL17 producing gamma delta T cells.
Thanking you
Rushikesh
By Life Technologies I have found the following antibody:TCR-gamma/delta Mouse Anti-Human mAb (clone SA6.E9), PE Conjugate. Could it help?
Please check these links for Cytokine Capture for enrichment of IL-17 producing cells
https://www.miltenyibiotec.com/~/media/Files/Publications/MACSandmore/MACS_more_12_1_2010.ashx
http://www.uwencode.org/proj/Science_Maurano_Humbert_et_al/data/TR_CD4_Tcell_isolation.pdf
Dear Rushikesh, I have tried to do the same experiment recently. You can perform the isolation of total TCRgd cells with the antibody that Bernd mentioned before conjugated to PE (for example) and then pull out labeled cells with Miltenyi anti-PE beads, that is how we do it. The problem is that TCRgd cells in peripheral blood are very inferquent, in the best cases they account for a 5% of total cells, so the amount of antibody and PE-beads you have to use is huge. Besides, I have checked IL-17 production in total PBLs and although I could perfectly see Il17 production by TCRab, I was not able to see IL-17 production in gated TCRgd cells. So I whatwe thought is that humal IL-17 secreting cells stay (or they are polarized in tissues) but they do not circulate. Of course to see IL-17 production you have to pre-stimulate the cells with plate-bound anti CD3 or TCRgd and the re-stimulate prior staining with PMA/iono + BFA, otherwise is imposible to see cytokine expression, so I do not think you can check directly on ex-vivo TCRgd cells cytokine expression with regular isolation/staining protocols. Probably the miltenyi IL-17 secretion assay that Korcan shows worth a try...
Hope it helps.
Dear All Thank you very much for all your suggestions. It is interesting suggestion to use IL17 secretion assay to isolate il17 producing gamma delta T cells. I had tried it once after isolation eventually I got only around 20x10^3 cells. Can you suggest me to expand this population to a sizable number to use them in assay without loosing their IL17 phenotype?
Thanking you
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