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T cell differentiation protocols


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#1 DWS

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Posted 25 February 2010 - 01:14 PM

I'm trying to differentiate various T cell sub populations from CD4+ and CD8+ cells magnetically purified from murine splenocytes. Can anyone provide some good protocols for Th1/Tc1, Th2/Tc2 differentiation? So far, I've had better luck with Tregs, but almost no luck with Th1/Th2. I'm plating the cells at 8x10^5/mL on plate-bound anti-CD3/anti-CD28 in 24 well plates. For Th1/Tc1, I've been treating them with IL-2 10 ng/mL, IL-12 20 ng/mL, and anti-IL-4 20ug/mL and incubating for 5 days before restimulation and harvest. For Th2/Tc2, I've been treating them with IL-4 (50ng/mL) and IL-2 (10ng/mL) for 3 days on plate-bound anti-CD3/anti-CD28 in 24 well plates. After 2 days, I remove them from anti-CD3/anti-CD28 and incubate for another 3 days before restimulation/harvest.

The problems I'm having are that the CD4+ cells in particular seem to do very poorly in culture and don't proliferate. The CD8+ cells seem to proliferate just fine. After 5 days I'm only getting about 7% CD4+ that are T-bet+ and 32% of CD8+ that are T-bet+ for Th1 differentiation. For Th2/Tc2, after 5 days I'm getting practically 0% CD4+ cells GATA-3 positive and only about 13% CD8+ cells GATA-3 positive. For Tregs, it hasn't been a problem with over 60% of cells becoming Tregs after 5 days. Does anyone have any suggestions? Thanks.

-DWS-

#2 miBunny

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Posted 25 February 2010 - 07:08 PM

To get a robust Th1 or Th2 differentiation, the cells need to be dividing really really well and it doesn't sound like yours are. I have never had as much luck with plate bound stimulation of CD4 T cells. Stimulating the T cells on irradiated rag feeders has always been the absolute best method. How much plate bound anti-CD3 and anti-CD28 are you getting? Are you seeing blasting cells? Are you refeeding them after you remove them from the plate?

I have found that feeding the cells every other day is best. Blocking antibodies are not required after the third day of culture. Just IL-2 and differentiation cytokine is enough. Your time line is very good (day 5 is optimal for Th effector development).

Some other tricks of the trade: use IMDM media and BD cytokines (Ebioscience cytokines have been hit or miss and R&D systems and peprotech have never really worked that well for me). I like the IL-2 that you can get from NCBI's reagent program. I have only used BD's blocking antibodies. The IMDM media brought my level of Th differentiation at least 10%.

Also... what is the backround strain of your mice? B6s are really really tricky to push into Th2.

#3 DWS

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Posted 01 March 2010 - 07:21 AM

Thanks for the help! I'm using 6 ug/mL of each for plate bound anti-CD3/anti-CD28. It seems to work well for me with Tregs. I am seeing blasting cells mostly from CD8+, but not really so much from CD4+. I haven't been refeeding them after removing them from the plate-bound antibody, but I wasn't refeeding for Tregs either. I will try feeding every other day. I am using RPMI, but I could try IMDM. Also, I am using peprotech cytokines, but they have always worked well for me in the past with other experiments and work fine for Tregs. I am using B6 mice which could also explain some of the difficulty, I suppose. What % conversion to Th1/Tc1 and Th2/Tc2 would be about average after 5 days if it is working well? With Tregs, I can get 60% CD4+ CD25+ Foxp3+ cells after 5 days and 20-25% CD8+ CD25+ Foxp3+ after 5 days.

To get a robust Th1 or Th2 differentiation, the cells need to be dividing really really well and it doesn't sound like yours are. I have never had as much luck with plate bound stimulation of CD4 T cells. Stimulating the T cells on irradiated rag feeders has always been the absolute best method. How much plate bound anti-CD3 and anti-CD28 are you getting? Are you seeing blasting cells? Are you refeeding them after you remove them from the plate?

I have found that feeding the cells every other day is best. Blocking antibodies are not required after the third day of culture. Just IL-2 and differentiation cytokine is enough. Your time line is very good (day 5 is optimal for Th effector development).

Some other tricks of the trade: use IMDM media and BD cytokines (Ebioscience cytokines have been hit or miss and R&D systems and peprotech have never really worked that well for me). I like the IL-2 that you can get from NCBI's reagent program. I have only used BD's blocking antibodies. The IMDM media brought my level of Th differentiation at least 10%.

Also... what is the backround strain of your mice? B6s are really really tricky to push into Th2.



#4 miBunny

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Posted 02 March 2010 - 06:58 PM

Tregs are a little easier to push than Th1 or Th2 (at least for me). I have had mixed luck with peprotech cytokines, some work well and some don't. I personally did not like them for Th assays but I've always had excellent luck with BD cytokines and largely good luck with eBioscience's.

CD8 cells activate easier than CD4. You can try taking the plate bound anti-cd3 and anti-CD28 to 10 ug. If you can irradiate splenocytes from a RAG KO mouse, you can add the stimulatory cytokines directly to the culture (no need to bind to the plate). This gives phenomenal activation and Th development.

For a B6 lymphocytes, Th1 development can go over 50%. Th2 development is usually quite a bit lower. On my best days, I could top 20%. With a Balb/C I could get pretty high Th2 development (30-50%)

#5 guppyde

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Posted 20 September 2010 - 06:29 AM

Hi, I'm going to work on lymphocytes differentiation in vitro. And would you please let me know how's your work on Th2 going on? Thousand thanks!! :)




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