Now I'm trying to analyze phosphorylation of signal molecules in Treg. To do that, initially I chose foxp3 staining kit from e-bioscience. But most of cytosolic proteins were gone, although foxp3 could be stained. Next, I fixed cells by 2% PFA @37C for 15min before permeabilization by above kit or True-Phos Perm Buffer from biolegend. We could see phosphorylation of signal molecules, but not foxp3...
If someone has experienced co-staining of foxp3 and phospho-protein or has an idea about that, please let me know.
Thanks
Have you tried Buffer kit from BD? FoxP3 and p-STATs can be stained at the same time from what they are saying.
http://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/immunology-reagents/immunology-buffers-and-ancillary-reagents/transcription-factor-phospho-buffer-set/p/563239
Helo!
I studied STAT5 phosphorylation in Tregs. I used BD CytoFix Fixation Buffer and BD Phosflow Perm Buffer. The FoxP3 expression and the Tregs percentage was slightly lower when BD buffers were used compared to eBioscience buffers. However, I was unable to detect STAT5 phosphorylation when I used eBioscience buffers; therefore, I recommend BD buffers despite the lower FoxP3 expression. Hopefully, I was helpful.
Hi, Yoichiro
Thank you for your response. I've not tried BD reagent. BD's data looks so nice, so I'll try using that!!
Best regards,
Ei
Hi Zuzana,
Thank you for your response.
As described above, I could not detect any foxp3+ cells using fixation with PFA and permeabilization with other reagent. Therefore, BD reagent which you recommended should be helpful for me, even though lower Foxp3 exression!
By the way, have you analyzed other phosphorylated protein in Treg?
Best regards,
Ei
Hi Ei!
Unfortunately, not yet. I am planning to analyse pSTAT1 and pSTAT3 in monocytes and dendritic cells, which is not easy to detect because some specific surface antigens (CD14, HLA-DR…) are sensitive to fixation/permeabilization procedure. However, I suppose that detection of other phosphorylated proteins in Tregs will be possible when the right buffers and protocol are used. I used Protocol II and III, BD Cytofix and Perm Buffer III. BD has a list of tested antigens and fluorochromes and recommended buffers and protocols on their website, which could be helpful.
Hi Zuzana,
Thank you for your reply. Fortunately, my friend has BD reagents which you recommended. So I'll try to analyze some phosphorylated protein in Treg using BD protocol. Thank you!
Ei
Hi Ei,
Beckman Coulter Perfix Expose Kit could be an alternative as well:
www.beckmancoulter.com/wsrportal/bibliography?docname=FoxP3+%20Tregs%20cells%20sensitivity.pdf
Greatings
Uwe
Hi Uwe,
Thank you for your information. Actually I didn't know that Beckman Coulter provided such a kit! I will try that as second choice.
Best regards,
Ei
Hi Ei. I am trying to look at pStat5 at basal levels in Tregs present in mouse spleen. Can you please share your protocol with BD cytofix and Phosphoflow perm buffer if that worked for your experiments. Thanks.
Atika Dhar
Hi Atika,
I just analyzed the phosphorylation of TCR proximal and downstream molecules in Tregs after the stimulation with anti-CD3/CD28 Abs, but not basal level. I don't know whether my protocol help you, but in my case, after the stimulation with Abs to CD3/CD28 (100ul volume), I add 1ml of Fix/Perm buffer to cell suspension ASAP. And then, according to the protocol of Transcription Factor Phospho Buffer Set (BD, 565575), I permeabilized and stain cells with Abs to Foxp3 and phosphorylated protein (1/10 scale).
To analyze the phosphorylation of protein on basal level, I think that you have to completely keep spleen on ice after isolation from mouse, and homogenize and fix cells ASAP.
If you find good protocol to analyze the phosphorylated protein on basal level, please share your protocol.
Thanks!
- Badges
- Science method