Multicolor flow cytometry is a powerful technique for the analysis of intracellular proteins that are expressed by specific cell types.
For many cell types such as Th17 cells, the combined use of cell surface and intracellular markers is necessary for definitive phenotypic identification. Simultaneous analysis of surface markers and signaling proteins can be used to characterize the nature of signaling responses within specific target cells.
While techniques for cell surface staining are relatively standard, optimal staining for intracellular markers often depends on the biology of the target protein. Depending on the protein’s location inside the cell, association with other molecules, and its stability, different cell preparation and staining methods are recommended. Cytokines, for example,are typically secreted proteins. However, if they are trapped inside the cell, they can be stained as intracellular proteins using protein transport inhibitors such as BD GolgiStop™ (containing monensin) or BD GolgiPlug™ (containing brefeldin A). Cytokines are relatively accessible using the gentle fixation and permeabilization afforded by BD Cytofix/ Cytoperm™ fixation and permeabilization solution.
In contrast to cytokines, transcription factors often are localized inside the nucleus and bound to DNA and other proteins. Phosphorylation of some proteins, such as Stat5, results in dimer formation that masks the phosphorylated epitope of interest. Also, intracellular phosphatases can quickly dephosphorylate these proteins. Therefore, after treatment, cells must be quickly fixed and subjected to stronger permeabilization conditions to allow the antibody to enter the nucleus and access the epitope within disrupted molecular complexes.
The permeabilization technique used can negatively impact the detection of cell surface and other intracellular antigens. The same techniques that allow access to the nucleus and open up DNA/protein or protein/protein complexes can often denature cell surface antigens, preventing their detection by antibodies. While detection of different intracellular proteins might require different conditions, the basic principles are the same: cells are fixed and permeabilized and then stained intracellularly with fluorescent antibodies.
Basic principles of intracellular staining
Cells are fixed and permeabilized (symbolized by dashed line membrane), stained, and then analyzed by flow cytometry. For studies of secreted proteins, cells are first treated with a protein transport inhibitor to allow accumulation of the target protein inside the cell..
BD Tools for Intracellular Flow Cytometry
To facilitate intracellular flow cytometry assays, BD has developed several kits, buffers, and protocols. In addition, many fluorescent antibodies specific for key cell surface markers have been tested in several buffer systems to save researchers' time, samples, and money. Commonly used BD buffers for particular applications include:
Detection of Cytokines
BD Cytofix/Cytoperm fixation/permeabilization solution (Cat. No. 554722) is suitable for staining most cytokines and cell surface markers. This buffer system can also be used in staining of some transcription factors and other intracellular proteins. This buffer system contains mild detergents along with a formaldehyde-based fixative.
BD Pharmingen™ transcription factor buffer set (Cat. No. 562574/562725) is designed for the staining of transcription factors alone or in combination with cell surface markers and cytokines. This buffer system contains mild detergents along with a formaldehyde-based fixative.
Detection of Phosphorylated Protein
BD Phosflow™ perm buffer III (Cat. No. 558050) is the recommended permeabilization buffer for phosphoepitope detection by flow cytometry. Perm buffer III is a harsh alcohol-based buffer. Alternative permeabilization buffers also are available to accommodate particular experimental requirements.
Optimal cell permeabilization conditions vary by epitope location
Optimal permeabilization conditions are determined by the accessibility of the epitope within the cell.
The phosphotyrosine 701 epitope (orange) is located within the Stat protein dimer, while the phosphoserine 727 epitope is located outside the dimerization regions. For the phosphotyrosine 701 site, a harsher buffer such as perm buffer III is required for staining. To induce protein phosphorylation, human peripheral blood mononuclear cells (PBMCs) were either left untreated (–) or were activated (+) with human IFN-α (Stat1 pY701) or PMA (Stat1 pS727). Cells were fixed using BD Cytofix™ fixation buffer and permeabilized using BD Phosflow™ perm buffer I, II, III, or IV, prior to staining with fluorescent phosphospecific antibodies.