Hello everyone,
Do you have experience in the isolation of Th17 cell clone from human PBMC?
One common method is to firstly isolate naive CD4+T cells, then to use flow cytometry sorting (CD161 CCR6), but the flow cytometry in my lab does not have the function of cell sorting.
Could you give me other suggestions?
Thank you so much!
Regards,
Junfen
I haven't run this kits personally but depending on your needs(Naive vs non-Naive) you can try several alternatives to flow. They can be pricey though:
Two step Magnetic Isolation (Positive selection of CCR6+, Depletion of nonCD4+,CD45RA-, CXCR3-): https://www.stemcell.com/easysep-human-th17-cell-enrichment-kit-ii.html
Magnetic Isolation of stimulated Il-17 producing cells (note you need to stimulate the PBMC to capture the cells and isolation based on CD45+IL17+): https://www.miltenyibiotec.com/US-en/products/macs-cell-separation/cell-separation-reagents/microbeads-and-isolation-kits/cytokine-producing-cells/il-17-secretion-assay-cell-enrichment-and-detection-kit-pe-human.html#130-094-542
If your goals allows you could also trying differentiating them yourself from a naive CD4+ T-Cell mix with stimulation, polarizing cytokine supplementation and neutralization of other differentiating factors(ie anti-IFN-g/anti-Il-4) . Though that would take quite sometime(7-10 days to differentiating) and will have to be optimized.
Hello William,
Thank you for providing so many useful information.
Actually before I have tried to use the combination of IL-1 and IL-23 plus PHA to induce Th17 cell differentiation from PBMC, but the results were not very good. I think later I will directly use naive CD4+T cells when the naive CD4+ T cell isolation kits arrive.
My final goal is to use Th17 cell in further co-culture. But I know PBMC can not be passaged, I can only add the poly-stimulis in the beginning of co-culture. Or do you have any suggestions?
Regards,
Junfen
Hi,
There is a kit to isolate these cells from PBMC. Its a bit pricey... but may be what you need.
The EasySep™ Human Th17 Enrichment Kit II is a two-step method designed to enrich for human Th17 cells from fresh peripheral blood mononuclear cells. First, CCR6+ cells are isolated by column-free immunomagnetic positive selection using antibody complexes and EasySep™ Releasable RapidSpheres™. Then, bound magnetic particles are removed from the cells and unwanted non-CD4+ T cells, CD45RA+ cells, and CXCR3+ cells are targeted for depletion using antibody complexes and EasySep™ Dextran RapidSpheres™. The final isolated fraction is enriched for IL-17-producing Th17 cells.
https://www.stemcell.com/easysep-human-th17-cell-enrichment-kit-ii.html
One note: I'm not sure of your PBMC source but if you can want to isolate the naive Th17 cells using one of the above kits it might worth using a leukphereis product(commonly obtained during platelet donation) or buff coat PBMC to increase yields.
For differentiation, you can re-stimulate the cells with polyclonal stimulus just to get enough cells for your needs. In general for these differentiation protocols the CD3/CD28 antibodies(either plate bound or on beads) are used instead of PHA the general consensus is that this is "cleaner". In addition you should consider adding other supplements such as TGF-B and Il-6. You could benefit from adding neutralizing antibodies(Il-4/IFN-g maybe others) against interfering cytokines as well. You can find clones that have been shown to neutralize in culture from one of the cheaper vendors like biolegend to keep costs down.
When working with whole PBMCs other cell populations may interfere with your differentiation so it is done on the CD4+ T cells. These T cells can be re-stimulated periodically and split as they grow so you should be culture for quite sometime (14-21 days or even longer) just don't centrifuge them. A note, most differentiation protocols seem to be 7-10 days though. Extended culture will also make cells less representative of in-vivo Th17.
If you decide to go with differentiation as your cell source for co-culture cells keep in mind that not all the T cells in your culture will be Th17 phenotype you can monitor this via flow.
Good luck!
Hello Jack,
Thank you for your suggestions.
I looked the information about the EasySep kit your recommended, but found the final percentage of intracellular IL-17+ cells is 9-16%. It is low for my experiment. Because I would like to directly use Th17 cells in my co-culture, it is better to get high purity of Th17 cells.
Regards,
Junfen
Hello William,
Thank you for your detailed information.
PBMC will be isolated from the blood pocket by Ficoll.
Regarding the effect of other cell types in co-culture, I want to firstly isolate high purity of Th17 cells, and then do co-culture with these Th17 cells. When looking at the two kits your recommended, I think IL-17 secretion Assay from MACS will be better. As finally the IL-17-secreting CD4+T cells have been enriched to 99%, that is shown by the company. Do you have any exprience by using this kit?
Actually I have another question. After Th17 cell differentiation by using polyclonal stimulis, can I collect these cells and freeze them for further experiments? Because in my co-culture, I need to seed MSCs firstly, after culture overnight, to add Th17 cells.
Thank you!
Junfen
Hello Jack Jack Chen ,
Have you ever tried this kit? If possible, could you tell me how many percentage of IL-17+ cells in CD4+T cells in your experiment?
Regards,
Junfen
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