I need your advise on the best way for the short-time T cells preservation in order to activate their proliferation and IFN-g production in 48h after isolation. Personally, I am familiar with T cell activation and have previously activated them with a-CD3- aCD28 beads as well as PHA, ConA or PMA/Ionomycin. All of those work perfectly on the freshly isolated lymphocytes. However, now we have to stimulate T cells 48h after isolation due to peculiarities of the protocol and we no stimulation in the positive control with a-CD3- aCD28 beads. PHA give us some proliferation though much less in comparison with expected rate. Briefly, we isolate healthy donors' PBMC by gradient centrifugation following CD14 MACS sorting. CD14+ monocytes then undergo differentiation into macrophages under several conditions during 48h.
Then we have to test their ability to inhibit (we are dealing with tumor-modified macrophages) T-cell proliferation - that makes the delay. We store lymphocytes (CD14 negative PBMC) on +4 in the MACS buffer. In 48h we have approx 60% live lymphocytes which we try to stimulate and co-culture with macrophages. Thus, the problem as I see it is the storage of the lymphocytes. Interesting enough, though our a-CD3- aCD28 beads stimulated control give no proliferation/IFN-g production, all macrophages (control and tumor-modified) are able to activate IFN-g production and/or proliferation of T cells...
You can indeed keep your PBMCs in complete medium in a CO2 incubator more than 48h with very little cell death but if you want to avoid any artefacts induced by such waiting period, you could alternatively freeze your PBMCs and thaw them when required after a minimum of 2h resting time in the incubator. We do that regularly with >95% viability. Let me know if you need more details on the freezing step.
Dear Alexandra,
do I understand you correctly that you store the T cells at 4°C (in the fridge) for 48 hours? I have never tried that, but I have done a lot of experiments quite similar to yours, when I differentiated monocytes to dendritic cells within 72 hours before using them to activate the autologous T cells left over from the CD14 selection (or plastic adherence). For these experiments, I just let the T cells sit in culture medium in the incubator for 72 hours (at rather high concentrations), and they worked fine, without much loss in viability and function. So I guess 37°C, 5%CO2 and culture medium containing FCS might be the far better solution than 4°C and MACS buffer.
Best wishes
Felix
I agree with sitting in the incubator in media. A critical issue for T cells is the right media. We have very good luck with Aims V, 10% FCS. If problems persist, it may be necessary to use human serum, and test the lots to determine which works best for your system.
Dear Alexandra, I agree with Felix. In addition I may suggest the negative selection of B cells. I may improve the response of T cells. Good luck.
Luiz
Dear Alexandra,
I´m also agree with Felix, I have worked with T cells, after a differentiation protocol, I realized INF-g measeurements and in my experience, you can leave them in the incubator with your usual medium (RPMI+ serum) but without activation (no CD3/28 or PHA) until you need it (I´ve never tried more than 48 hours), I never saw loss of functionality or decrease in INF-g.
Good luck!
Alexandra, those PBMC should remain in regular supplemented medium as RPMI 10% FBS and antibiotics, in incubator at 37°C 5% CO2. Other thing is that working with human PBMC or T cells you shouldn't use ConA because ConA is a good mitogen for mouse T cells. Good luck
Dear Alexandra, try to use storage at RT in the dark. Resuspend isolated cells in complete medium (RPMI + 5% human serum + L-glutamine + 2ME) in the flask, tightly close the lid and place the flask into a drawer of your table. This procedure is working well even with mouse cells, which are much more delicate.
Agree with what was said above. T-cells/PBMCs have to be kept in a 37oC incubator. As a medium, you can use either AIM-V, X-VIVO15, Biotarget, or even RPMI... with either 10% FCS or 5% HS (and with some 50-300 IU IL2). Good luck
thank you all so much, guys! so, RPMI (we use it for all further procedures) and 10% FCS and will keep cells in the incubator. Thank you all again!
Dear Alexandra,
I agree with the others that media (RPMI, ultraculture, AIMV...) should be taken. depending on the type of experiment addition of IL-2 (20-100 IU/ml) is a good option.
Best wishes
Thomas
I also agree with Flex. I would also like to add that I used to use DMEM with FCS, P/S and AB+ve serum. It is best to keep the cells in incubator and in U bottomed tubes. Depending upon type of experiment IL-2 can be added. Hope this helps.
Best wishes
swe
I disagree with above. I would store them in hypothermosol solution at 4 d C. This is buffered and prevents sheer stress, activation, attachment.
You can indeed keep your PBMCs in complete medium in a CO2 incubator more than 48h with very little cell death but if you want to avoid any artefacts induced by such waiting period, you could alternatively freeze your PBMCs and thaw them when required after a minimum of 2h resting time in the incubator. We do that regularly with >95% viability. Let me know if you need more details on the freezing step.
Thank you, Damien. I am going to try incubation. If I will not be satisfied with results I will try to freeze cells and will contact you.
Alexandra, I beleive 37degC/5%CO2 incubation which many people mentioned must work fine. It is described in T cell reactivation protocols where you keep activated T cells for couple of days in the plain FBS-cotaining medium before subjecting them to reactivation. I know, my friend did it this way.
Holding mouse T cells for even a short time on ice or at 4oC induces apoptosis within 24-48 h. Human cells are a bit sturdier but I agree with all of the above - incubator or even RT is preferred.
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