Successful simultaneous measurement of cell membrane and cytokine induced phosphorylation pathways [CIPP] in human peripheral blood mononuclear cells.
Phenotyping and simple enumeration of peripheral blood mononuclear cells (PBMC) is of limited value for the assessment of many clinical states. As a preferred alternative, cell surface phenotyping may be combined with functional assays for enhanced assessment of altered cells circulating in patients. One simple, yet informative and rapid approach is to examine signaling within individual cells following brief periods of stimulation via flow cytometry. Although monocytes and lymphoid cells can be distinguished based on size, current permeabilization strategies necessary for identifying intracellular phosphorylated signaling molecules largely compromise the labeling of cell surface proteins used to distinguish individual cellular subsets. We have successfully developed conditions that allow for simultaneous detection of cell surface proteins and intracellular phosphorylated proteins in human PBMC following rapid in vitro cytokine stimulation. We analyzed permeabilized CD4, CD8, CD14, CD19, and CD56 expressing cells together with intracellular pSTAT1, pSTAT3, pSTAT5, pSTAT6, pp38 MAPK, or pERK1/2 within total PBMC. Of the permeabilizing conditions tested, 75% methanol enabled superior simultaneous detection of both cell surface and intracellular epitopes. This method enables the rapid functional analysis of subsets within complex cell mixtures and provides an opportunity for assessing abnormalities arising in the setting of acute or chronic inflammatory states.
Montag
David T
DT
Department of Bioengineering, University of Pittsburgh, PA 15219, USA.
Lotze
Michael T
MT
D000096:Acetone / Q000494:pharmacology; D015703:Antigens, CD / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis*; D002463:Cell Membrane Permeability / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D016753:Interleukin-10 / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000432:Methanol / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D050791:STAT Transcription Factors / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D015703:Antigens, CD; D000954:Antigens, Surface; D016207:Cytokines; D016898:Interferon-alpha; D007376:Interleukin-2; D050791:STAT Transcription Factors; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D012503:Saponins; D016753:Interleukin-10; D000096:Acetone; D015847:Interleukin-4; D007371:Interferon-gamma; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D000432:Methanol