Dear all,
can sombody give me some suggests, I do not why I can not detect IL17A Expression, I really do not know what is wrong with my differentiation, please give me some advise.
I isolated Naive CD4 cell using Biolegend kit (the purity is around 80%), then using 5 ug/ml anti-CD3 coated 96-well-plate for 2 h in the incubator, wash with PBS for two times, each well I add 2*10^5 cells.
For Differentiation condition:
5 ug/ml anti-IFN-g, 5 ug/ml anti-IL4, 20 ng/ml IL6 and 0,5 ng/ml TGF-b, 1 ug/ml soluble anti-CD28, total 200 ul medium with cytokine in each well, at Day 2 I will suspend cells and replace to a new 96-well-plate, at Day 3, I restimulate cells with TPA/Ionomycin, and after 2 h add Glogi-stop and Glogi-plug for another 4 h, the did FACS. some time I can detect IL17A, but not two much, maximum 20%, but this time there almost no IL17A Expression, but restimulation and FACS staining have no Problem, because I have Th1 cells, it works. so my question is what should I do to make Th17 differentiation better, and what is the problem with this Experiment.
This was the Th differentiation protocol I used and it worked well for me. BTW, I did my PhD in Wurzburg.
"Lymph nodes and spleens are obtained from mice. The tissues are ground and the cells are then treated with an RBC lysis buffer to remove the red blood cells. The cells are then passed through a cell strainer to obtain a single cell suspension. The naïve CD4+CD62L+ cells obtained from MACS are resuspended in RPMI supplemented with 10% FCS, penicillin / streptomycin, glutamine, HEPES and sodium pyruvate. Differentiation is done on 24 well plates. 0.5-1 X 106 naïve T cells are plated out per well. The cells are then stimulated with 5µg/ ml anti CD3 and 1µg/ ml anti CD28 in the presence of cytokines. The cytokine concentrations used for are as follows: Th17: TGFβ- 2ng/ml, IL-6- 30 ng/ ml, IL-23- 15 ng/ ml, IL-1β- 10 ng/ml, aIFNg- 1µg/ ml; Th1: IL-2- 2 ng/ ml, IL-12- 10 ng/ml, aIL-4- 1µg/ ml. The cells are cultured for 5 days. On day 5, the cells are replated in restimulation media (RPMI supplemented with 10%FCS, penicillin/streptomycin, glutamine) and restimulated with ionomycin (750ng/ml) and phorbol- myristateacetate (50ng/ml) for 4 hours in the presence or absence of Brefeldin-A (5 µg/ 97 ml). The cells restimulated in the presence of Brefeldin-A are used for intracellular cytokine staining."
None of the protocols in our lab used anti IL-4. I can't remember what percentages of IL-17+ cells I used to get, it was so long ago. I looked up conversations that I had with one of my colleagues in 2013. He mentioned that switching from 5% serum to 10% serum made a huge difference. I did mention to him that I had better luck measuring IL-17 in ELISA than in Flow cytometry. But this was 8-10 years ago and the technique has become much better. Also, we change media for re-stimulation and I seem to have had better results when cells were plated with fresh re-stimulation media. I checked my paper and seems I only had ELISA data in there ( https://www.jimmunol.org/content/188/1/216.long ; Fig 2A).
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