Dears
I need to activate CD8 T cells of OTI mice by OVA peptide.
according to this paper (Pulle, G., M. Vidric, and T. H. Watts. 2006. IL-15-dependent induction of 4-1BB promotes antigen-independent CD8 memory T cell survival. J. Immunol. 176: 2739–2748.) I culture 3X106 of CD8 T cells isolated by magnetic beads from OTI mice with 6X106 mitomycin C treated B6 splenocytes per ml and 3ug OVA for 20h. after 20h I wash cells and resuspend them in fresh medium 106/ml and culture them for next 24 h. after that I wash cells again in day 2 and resuspend them in fresh medium and cultured them for next 24h. the problem is although cells look very fine in the culture at day 2, at day 3 many of them look changing appearance and become small and dark. I think they are dying. Can it be for too much washing? any other reason?
I need a protocol to generate a high number of activated OTI cells that be transferred into host mice and survive there and have a good Cyrotoxic activity.
any suggestion is highly appreciated.
Thank you very much
Hi,
Usually, it is better to use BMDC to activated the CD8 in vitro + cytokines.
They are several protocols.
Look those 2 papers, they have activated CD8 in vitro and test cytotoxic activity in vivo.
http://www.ncbi.nlm.nih.gov/pubmed/18825745
http://www.jimmunol.org/content/177/5/2976.full
Good luck.
J
Thank you dear Joao and Dimitri
Your answers were very helpful.
Thank you
Masoud
Dears
I came back with the answer,
I tried different protocols and finally this one works very well:
1. First make a cell suspension of splenocytes in the medium. Then cells were plated (day 0) at 5 × 106 per well in RPMI medium (PAA Laboratories) containing 10% heat-inactivated fetal bovine serum (Sigma) and supplemented to a final concentration with l-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), 2-mercaptoethanol (50 μM) This 2ME is very important! (1ml/well of 24 well plate). Before seeding cells in wells add 2ug/ml OVA peptide (OVA 257-264)
2. After 24h incubation add 2ml fresh medium and then after pipetting divide cells into 2 new wells. That means if you started with one plate now you have 3 plates.
3. Incubate them for next 24h. At day 2 add 1 ml fresh medium to each well and after pipetting divide cells into a new well. So now we have 6plates.
4. incubate another 24h. At day 3 cells are ready. Usually, 2 plates at day 3 totally have around 108 activated OTI. do not worry for other cells because most of them are already dead and your culture is almost only OTI cells.
5. wash them 5 times by 50ml PBS. Cells are ready for transfer.
Thank you
Masoud
Dear Masoud ,
I had the same problem druing last 3 weeks , I hope that your protocol would be fine for our CD8 cells from OTI as well . I tried with two diffrenet concentration of OVA peptide (OVA 257-264) 1uM/ml also with 1nM/ml. Did you add 2ME with fresh medium each day ?
Kind regards
Farahnaz
Dear Frahnaz
I solved that problem.
Instead of adding peptide into the medium, I pulsed the total cells with 2ug/ml OVA peptide for 4 hours. Then cells must be washed extensively and finally resuspend in complete medium (5milion cells/ml/well) in 24 well plate. Then the continue is as I mentioned in the original question.
After three days activated cells with a purity of 90% are ready for injection or in-vitro experiments.
Medium is fresh R10 medium.
I hope it helps
Masoud
Dear all,
a bit unrelated, but recently I got to know from a colleague that OT-I cells can be activated by only applying the SIINFEKL peptide. I haven't found literature on this. I used 1.5x106 cells in 1 ml medium and added SIINFEKL at 1ug/ml. I incubated overnight and obtained increased IL12RB2 expression and when incubating for 3 days I saw proliferation. This was a quick assay and I did not measure cytokines, as I was just interested in having some lysates to test IL12RB2 antibodies for WB. Does this mean that spontaneous activation can occur without MHC and costimulation?
Hi Masoud Akbari,
Thanks for sharing good protocol and details.
I need few suggestions.
(1) How do you pulse the cells with ova peptide?
Any instrument or some protocol
(2) Any need of CD3/CD28 and IL2 in the process?
(3) What will be the best time to add ova if we are unable to pulse the cells?
After the initial 24 hours or at 0 hours when the splenocytes culture will be started.
HI, i am wanting to look to at OVA peptide presentation in mouse DC and use OT1 and OT2 mouse T cells. I have tried hybridoma T cell specific for OVA peptides, but want to try primary T cells, so for OT-1 and OT-2, I plan to load DC with OVA and then add the OT-1 or OT-2 T cells and do ELISPOT for IFN or IL-2. My question is do I need to activate the T cells after purification , if so how> anti-CD28 etc, does anyone have a protocol they can share>
thanks
Masoud Akbari Hi Masoud, I tried the method you mentioned previously, it works well. But somehow the T cell proliferation is not that good. I can only get 4-5x10^6 T cells after 4-5 days of culturing starting with 1/3 of the spleen. I do see cell is proliferating and the T cell killing in vitro is working too. What do you think the possible reason is? Do you do red blood cell lysis before pulsing? Thank you!
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