Looking to use phospho-antibody (Ser177) for p-eNOS staining by flow cytometry in human umbilical vein endothelial cells.
Hi Dinesh Kumar Parandhaman ,
I am struggling to do pAKT expression on PBMCs for sometime. As you mentioned above, may i ask you for the protocol of the same?
Anticipating your response.
Thanks
Hi Mark,
Yes, I've used antibodies for pSTAT3 and STAT5 with flow cytometry. We used the BD Cytofix and Phosflow Perm Buffer III to fix and permeabilise before staining. Hope that helps!
I have done. Akt signaling in pbmcs. You have to check flurochrome compatibility before using the buffer recommended by Vanessa. Because perm 3 contains methanol. It can bleach the flurochromes. You can view the compatibility list either from Biosciences or bd
Hi Mark,
I have done extensive phospho-protein staining in mouse and human B and T cells for Syk, Erk, p38, BTK, PLC-gamma II and STAT5. I would suggest using FoxP3 staining buffer produced by eBiosciences for fix and perm step. This buffer preserve the surface markers reasonably well and generates very good signals for intracellular phospho-protein. While using this buffer set, please follow manufacturer's instruction.
Specific detail about the buffer is as follows:
eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set.
ThermoFisher Catalog number 00-5523-00.
URL: https://www.thermofisher.com/order/catalog/product/00-5523-00.
Hope you will find the information useful.
Good luck!
Best regards,
Mrinmoy
Hi Dinesh Kumar Parandhaman ,
I am struggling to do pAKT expression on PBMCs for sometime. As you mentioned above, may i ask you for the protocol of the same?
Anticipating your response.
Thanks
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