18 replies to this topic

[kalgopa]

zodiac1505, on Oct 2 2009, 05:24 AM, said:

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.

You may want to look at this for the summary of C57Bl6 TH17 differentiation protocols:
http://knol.google.com/k/kalpesh-patel/th1...vni7y960bgc1/5#

[tar bal]

zodiac1505, on Aug 25 2009, 03:14 AM, said:

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.

I was also wondering, along with another poster, the specifics of handling the plating and medium changes.

I have read protocols where medium changes and cytokine addition occur on day 2 and 4 (day 0 being plating day), but I cant't figure out how this can be managed on a plate larger than 96 wells without losing cells (and beads) in the spins and generally creating a lot of cell handling mess.

I was hoping to do this in a 48 or 24 well tray, because by the time I get through with intracellular staining and all the spins, etc, I loose a lot of cells if my source is just 1 well from a -96 well tray. Generally, I split the 1 well on day 4, then recombine it for the intracellular staining. It's still not enough. Seeing the pellet get smaller and smaller with each successive spin during permeabilization and staining is depressing!

The 24-well protocol referenced in the previous post is a 3-day with no re-feed. I am doing a 5-day human cell protocol, so I need to re-feed and split. Any suggestions?

Thanks so much, I appreciate the time taken by folks to help out.

Edited by tar bal, 27 May 2010 - 08:33 AM.

[oncology]

Hi all, me new to this Forum, I have recently tried for Th17 differentiation, after many trails I succseed upto 10% from total splnocytes, witout any purification, because I am polarizing memory Th17 cells, which mice was 3 times vaccinated with my TAA in my studies, now the problem bothering me is Th1 population(IFN-g positive cells) in Th17 Polarization condition.

I am Polarizing Th17 antigen specific cells (3-vax with anitgen) in vitro stimulation for 7-days
antigen 10ug/ml
il-2-U/ml
IL-23 50ng/ml
anti-IFN-g 10ug/ml
in RPMI-10% for 7-days ....after intracellular staining I got 10% of Th17 cells. and 21% IFN-g positive cells which i dont want there.

for Th1 polarization, I am using IL-12 20ng/ml
anti-IL4 10ug/ml..but any getting only 16% IFN-g positive cells gated on CD4.

any plz help me some suggestions..

someone suggested me Th17 (IL17)secretion assay kit from Miltenyibiotec...to purify cells....is it good to use before or after polarization,,can we use this purified population for adoptive transfer expt in vivo???????

thanks in advance for suggestions and help.

[Simha]

Hi ,

I am new to this group. I am searching for a human cell line that can consistantly express IL-17. So that I can screen my drugs for their inhibitory activity.

My Literature search did not reveal me any such human cell line availailty. Most of the search made me understood that we have to isolate naive CD4+ T cells , and then make them polarize to Th17 cells. Later these Th17 cells upon some stimulation from cytokines expresses high leveles of IL-17.

Can you please suggest me the best experimental plan of studying IL-17 expressions / production by an ELISA.

Thank you in advance.

Simha