I want to study intracellular signaling in mouse hematopoietic stem/progenitor cells. Due to the fact that stem/progenitor cells are a very small population, it would be cool to use phosflow (detection by FACS) - especially the combination with surface marker staining would be very interesting for me ! Does anybody have good protocols - especially for hematopoietic cells?
I agree- CyTOF is the way to go for looking at multiplex phospho analysis coupled with flow cytometry. There are mass labeled antibody panels being developed by different companies for the platform (such as BioLegend). It is offered commercially by DVS Sciences.
Alternately- for a fluorescence based flow, look at Cell Signaling's offering of antibodies along with Epitomics. At least some of Cell signaling's offerings are already conjugated.
I agree, the multiparametric flow cytometry-based assay is good for your aim.
Here is an example:
http://onlinelibrary.wiley.com/doi/10.1002/cytob.21125/abstract
If you ask this question, then I don't recommend starting with mass-spec cytometry. Start simply and work your way up. Pay attention to sample handling prior to fixation. Methods papers are Fleisher & Holland Clin Immunol 1999, 2001; Chow & Hedley: Cytometry 2001, Perez & Nolan: Nat Biotechnol 2002, Methods Mol. Biol 2004; Krutzik & Nolan: Cytometry 2003; Krutzik & Nolan; JI 2005; Schulz & Nolan Curr Protoc Immunol 2007; Jacobberger etc Cytometry 2003; Chow & Shankey: Cytometry 2005, Curr Protoc Cytom 2008; Woost et al. Blood 2011 (not really methods, but has a detailed protocol in supplement). Unless experiments eliminate it, for starting out, I recommend Chow's whole-blood approach.
If you are starting out, then I recommend you get in touch with your BD rep. They are the company that has the widest variety of phospho-specific antibodies, and they want to sell them to you so they will invest time in helping you to design your experiment. They also have good information about what fix/perm conditions work best for which antibodies, and how to couple it with surface antigen staining. But seen as you will need to use a relatively large panel of surface markers, you will likely need to do some iteration of the Stain-Fix-Perm protocol (http://www.bdbiosciences.com/documents/Phosflow_Alt_Protocol2_Fix_Stain_Perm.pdf)
Good luck.
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