Hello,
I am working on freshly isolated plasmacytoid dendritic cells. I stimulate them with Imiquimod (TLR-7 agonist) to see the phosphorylation of akt (ser473) after 90 min by flow cytometry...but any how I am not able to detect phosphorylation...so I am not understanding whether my stimulation agent or stimulation time or staining procedure is going wrong somewhere...can serum starvation of cells be helpful for this?
Can anyone guide me regarding this?
I tried it once with flow cytometry, but I got much better results using Western Blots (using CellSignaling ab). In my cells (moDC), I got the strongest signal 20 - 40 min after stimulation with LPS and lost the signal between 60 and 90 min after stimulation. I got also a pretty good signal by using GM-CSF and IL-4 alone.
I think your problem is, that you wait way too long. Most signaling events occur really fast (1 - 30 min after stimulation) and decrease rapidly. See also: http://www.bdbiosciences.com/documents/Suggested_Stimulation_Conditions_for_Phosphoprotein_Detection.pdf
You should try to detect your signal 20 min after stimulation.
Additionally, buffer conditions are also very important. See: http://www.bdbiosciences.com/documents/Phosflow_Alt_Protocol2_Fix_Stain_Perm.pdf
Good luck!
For pDC stimulation you can use with CpG A. This is also a TLR 7 agonist but I am not stating that you can not use imiquimod.
may be the following link can help you.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2271003/
Freshly isolated pDCs are often obtained by purification with MACS CD303 or CD304.
If this is your case, be careful because the antibodies used for the purification are strongly activator (or inhibitors).
Prefer depletion methods to avoid this inconvenience.
For my part I use antibodies that are not active including CD303; for CD304 or CD123 is a bit more complicated.
Pay attention also to contamination by basophils that are not eliminated by DC304 and CD123, and after degranulation will have an unexpected effect on pDCs.
For the activation of pDCs, CpG are actually very effective: choose the good CpG.
Otherwise Imiquimod (or Resquimod) are also active; R848 seems most appropriate.
Thank you to all of you for your valuable suggestions... @ michael....can you please send me detailed methodology of western blot used for detection of phopshorylation of akt? My concern is low number of pDCs...how much pDCs will be needed for cytoplasmic extraction and detection of p-akt? what are the buffers used for extraction? Is addition of phosphatase inhibitors are absolute necessary?
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