18 replies to this topic

[zodiac1505]

Hi

I have been trying to do some Th17 differentiation for a long time. I use an established protocol from another lab and start with naive T cells. After restimulation, I tried intracellular staining with IL-17/ IFN or with isotype controls. No matter what I try, I have never been able to detect more than 1% of cells which are IL-17 positive. Its the same case with IFN. The isotype controls as well show similar results. I tried ELISA and CBA on media from the cells and got really high signals for IL-17. Anyone has got any suggestions on what to do?

Thanx

[miBunny]

T helper differentiation protocols can be really tricky!
Can you please provide a detailed protocol?    what type of cells (mouse or human and if mouse what strain of mice)
How are you activiating?  Are the cells really blasting?
How long are you differentiating?
How much cytokines are added (are you adding neutralizing abs)?
When are you restimulating?  and how? are you adding golgi block or goldi stop?
How long are you restimulating?

[zodiac1505]

miBunny, on Aug 25 2009, 01:54 AM, said:

T helper differentiation protocols can be really tricky!
Can you please provide a detailed protocol?    what type of cells (mouse or human and if mouse what strain of mice)
How are you activiating?  Are the cells really blasting?
How long are you differentiating?
How much cytokines are added (are you adding neutralizing abs)?
When are you restimulating?  and how? are you adding golgi block or goldi stop?
How long are you restimulating?

Hello

I usually use NOD CD4+CD62L+ cells sorted by MACS. I usually pool the T cells from LN and Spleen. I use APCs. I then add various combinations of cytokines to the cells the same day that I plate the cells out. I have tried just with aCD3, or aCD3+TGF, aCD3+TGF+IL-6, aCD3+TGF+IL-6+IL-23, aCD3+TGF+IL-6+IL-23+IL-1b. I then leave the cells for 4 days. On the 4th day I restimulate the cells for about 4 hours by adding PMA and ionomycin. Depending on whether I am doing intracellular staining or ELISA, I add or do not add golgiplug (BD Biosciences). I don't add neutralizing antibodies. The cells seem to be blasting ok. Hope I answered all your questions.

[miBunny]

I would suggest adding in anti-CD28 (1 ug/mL) and IL-2 (2-10 units/mL of human or 2-10 ng/mL of mouse IL) to your initial stimulation conditions.

Keep your differentiation cytokines at 10 ng/mL (I found that higher concentrations gave decreased T helper development for all subtypes).  Feed the cytokines every other day.  If the cell media turns yellow on an a non feeding day, just add more media (w/o cytokines).  

Neutralizing antibodies can really help.

Keep the cell density at 1-5 million per mL.

What is the source of APCs?

The best day for restimulation in my hands was day 5 (if stim was day 0).  The percent cytokine positive cells gets lower with each passing day after day 5.

Are you adding the golgiplug at the same time as the PMA/Iono?  I usually add PMA/Iono for 2 hours, add golgi plug (or stop) and then stimulate for an additional 4 hours.

Are you seeing good T cell clumps?  After 2 days or so, the T cells should be nice and clumpy.  Don't break up the clumps.

I typically see 20-30% Th17 from a B6 mouse.

[zodiac1505]

miBunny, on Aug 26 2009, 03:29 AM, said:

I would suggest adding in anti-CD28 (1 ug/mL) and IL-2 (2-10 units/mL of human or 2-10 ng/mL of mouse IL) to your initial stimulation conditions.

Keep your differentiation cytokines at 10 ng/mL (I found that higher concentrations gave decreased T helper development for all subtypes).  Feed the cytokines every other day.  If the cell media turns yellow on an a non feeding day, just add more media (w/o cytokines).  

Neutralizing antibodies can really help.

Keep the cell density at 1-5 million per mL.

What is the source of APCs?

The best day for restimulation in my hands was day 5 (if stim was day 0).  The percent cytokine positive cells gets lower with each passing day after day 5.

Are you adding the golgiplug at the same time as the PMA/Iono?  I usually add PMA/Iono for 2 hours, add golgi plug (or stop) and then stimulate for an additional 4 hours.

Are you seeing good T cell clumps?  After 2 days or so, the T cells should be nice and clumpy.  Don't break up the clumps.

I typically see 20-30% Th17 from a B6 mouse.

I usually use 0.5- 1 million T cells and 2.5-5 million APCs per well. I add 2microg aCD3, 2ngm TGF, 30 ngm IL-6, 15-30 ngm IL-23 and 10 ngm IL-23 per well. I add them the same day as I plate the cells out and restimulate on the 4th day. I add golgiplug the same time as PMA/ ionomycin. I usually use the CD4- cells from MACS (spleen and LN pooled) as APCs. There were papers saying that probably Cd28 co stimulation or the addition of IL-2 can reduce Th17 differentiation. Does the addition of neutralising antibodies help a lot? I can see clumps of cells but I am not sure whether its less than what it is supposed to be. I am planning to try out your protocol but if you think the antibody concentrations I use could be the problem, could you let me know which ones I should increase and which one I should reduce? And also which medium do you use? I used to use RPMI but then there was a paper suggesting that IMDM could be much better. And I tried it and got the same feeling. So I now use IMDM.

[miBunny]

I found IMDM gave better Th development than RPMI or high-glucose DMEM.  It is formulated to support highly proliferative cells and I found that in a head-to-head comparison that it gave better Th1 and Th2 development.  This was done pre-Th17 days so this wasn't thrown into the mix.

Neutralizing antibodies can help a lot.  You really only need them for the first couple of days.  They don't need to be added after day 3.  I get them from BD (low endotoxin/no azide version) and use them at 1 to 1000 (should be 1 ug/mL).

I always threw IL-2 and CD28 in the mix.  Hmmmm.... its called path dependency... I always set up Th cultures this way and didn't think about it. Might need to do some reading!

We ( my bench mate and I) tested the PMA/Iono + golgi stop (added at different times after stimulation).  It really helps to give the cells 2 hours to activate.  We both found that the percent responding cells dropped quite a bit if the golgi stop was added at the same time as the P/I stim.

Is your media turning yellow by day 3 or 4?  At the concentration of cells you have, you should be going yellow by then.  Have you counted the cells (just count 1 well as a marker-I found that breaking up the cell clumps seemed to decrease how much Th development there was).  This should give you an idea of how well the cells are proliferating.

[zodiac1505]

Thanx for all the suggestions. You are right, my media does turn yellow. I have always left it like that. I am planning to do a differentiation following all your advice probably next week. Will let you know of the outcome. Then how do you usually do intracellular staining? I use the intracellular staining kit from BD. And I stain the cells either with IL-17/ IFN mix or isotype mix. The isotype always give staining quite similar to the Ab mix. Maybe there is smething wrong with the staining. I tried increased Ab conc last week. But couldn't see any improvement.

[miBunny]

If your media is yellow, you need to add more. This indicates the pH has gone acidic which makes the cells unhappy

There are two potential issues.
1. You are not getting Th17 development
2.  Your intracellular stain is not working.

You might want to try stimulating some cells and collecting the sup at 48 hours to look at cytokine production by Elisa or bead array (BD may have Th17 CBA by now- and you can stretch those kits 10 fold-if you are curious ask!)

Good luck!!  My fingers are crossed!!!!!

[zodiac1505]

Could you please let me know how you used to do Th1 differentiation? I want to do it along with the Th17 differentiation. Have been looking around in papers but it might be better to ask someone who has done it  

[miBunny]

Th1 is the easiest!
anti- CD3 and CD28, IL-2 (10 Units), and IL-12 (10 ng/mL), anti-IL-4 (1 ug/mL) on day 0,

Day 3:  IL-2, IL-12 (10 ng/mL), anti-IL-4 (0.1 ug/mL)

Day 5: IL-2
Day 6 or 7: restimulate for cytokines

On "even days"if the media is too yellow, I add more ( no cytokines).  I found T helper development decreased if cytokines were given every day.

IL-12 and anti-IL-4 comes from BD.  I've had the best luck with BD's cytokines.  Like all BD products, you pay more but generally get really good stuff

[zodiac1505]

Thank you so much. I am going away this week, so will most probably be doing the assay next week. Will update you after that   And I had one more doubt abt the intracellular staining. I found procedures where they add gogiplug to the buffers, staining solutions and wash solutions while doing the staining. I have never done this. Just wanted to check whether I should add golgiplug the whole way until FACS or is it enough to use it just while restimulating.

Edited by zodiac1505, 09 September 2009 - 12:15 AM.

[miBunny]

Wow, that sounds like a waste of golgiplug!  we've never done this...

Once the cells are on ice, the metabolism will be so slowed that the golgiplug shouldn't be necessary.  That should only be 30 minutes and then the cells should be fixed.  Once the cells are fixed they aren't going to need golgiplug!

Have a nice time off and good luck next week!!!!!!  My fingers are crossed!

[zodiac1505]

Sorry about the late reply. Had a busy 2 weeks. I tried out the Th17 protocol you suggested. It works great As usual, the intracellular staining doesnt work. I still didnt see a population of Th17 cells. But ELISA shows that tons of IL-17 is produced. My old protocol gave like 1-2 ngms of IL-17 / ml. But now they give 10-12 ngms/ml. So I guess it should be working. But my media still doesnt turn yellow. I hope that doesn't matter. Th1 differentiation seems to work as well. So glad I found u    Thanx a lot.

Edited by zodiac1505, 02 October 2009 - 04:24 AM.

[miBunny]

That is awesome!!!!!!!

[venuste]

Hihi,

I am trying to differentiate Th17 cells from Treg and came across to this post. Just wondering which plate would you suggest? 96 or 24?

Thanks!

miBunny, on Oct 6 2009, 06:54 PM, said:

That is awesome!!!!!!!