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Intracellular Cytokine Staining
What is Intracellular Cytokine staining?
With increased interest in cytokine regulation of cellular growth and differentiation, many investigators are turning to methods for analyzing cytokine production. Most methods involve measuring the amount of secreted cytokines present in serum or supernatant. In addition to requiring longer activation times, these methods do not enable the investigator to determine the phenotypes of the cytokine-producing cells. Intracellular staining system measures the expression of cytokines within cells. The aim of intracellular cytokine staining is to make use of the rapid flow cytometric analysis of cells to provide both surface phenotype and intracellular cytokine content of single cells within a population of cells responding to antigenic or mitogenic stimulation. The technique initially requires fixation of cells in paraformaldehyde, which cross-links proteins and prevents their loss through leakage. Secondly, permeabilization of cellular membranes with a detergent generates gaps in the membrane, allowing specific cytokine antibodies access to the cells' interior.
Advantages of this methodology.
Intracellular cytokine staining of cells and subsequent flow cytometric analysis can yield rapid and specific information on the cytokine production properties of individual cells within a mixed population of cells. The time from activating the cells to interpreting the data can be as short as 5 hours. Being able to look at individual cells and identifying their phenotype as well as their cytokine profile has tremendous benefits. This techniques circumvents the need to isolate populations of cells by physical means such as panning methods, columns, density gradients, etc., which rarely yield populations of cells of sufficient purity that allows for easy data interpretation.
Principle of the test.
Stimulated peripheral blood mononuclear cells (PBMCs) are fixed, surface stained and then permeabilized, allowing conjugated antibodies access to proteins within the cell. Cells are initially subjected to an activation in the presence of Monensin or Brefeldin A (BFA) which inhibit intracellular transport of proteins so cytokines produced during activation will be retained inside the cell. A following fixation step is necessary in order to minimize leakage of proteins out of the cell before allowing the conjugated antibody to penetrate and bind to its target cytokine within the cell. Following a final wash, the cells are analyzed on a flow cytometer. Flow cytometric analysis of carboxyfluorescein conjugates will generate a signal which can be detected in the FITC signal detector (usually FL1) while R- phycoerythrin conjugates will generate signal which can be monitored by the detector reserved for phycoerythrin emission (usually FL2).
Cell activation and fixation
PBMCs of healthy donors are separated via Ficoll-Paque density-gradient centrifugation. We use standard techniques and resuspend PBMCs at 2x106 cells/ml in RPMI-1640 with 10% heat-inactivated fetal calf serum. For stimulation, Phorbol 12-Myristate-13-Acetate (PMA; final concentration 25 ng/ml) with Ionomycin (final concentration 10µg/ml) is utilized. Activation is performed in the presence of BFA or Monensin to retain cytokines inside the cells. Then cells are fixed by 1% paraformaldehyde (PFM) in PBS.
Selection of antibodies
Intracellular staining antibodies are selected for multiparameter flow cytometric analysis of cells. To stain for surface proteins, such as CD3, CD4, CD8, etc., we determine whether the surface protein is adversely affected by the fixation and permeabilization steps. Should this be the case, surface staining of cells prior to fixation and permeabilization are performed. For intracellular cytokine staining, the cells must first be fixed then permeabilized.
Permeabilization
For permeabilization, 0.1% saponin in a balanced salt solution is commonly used. However, It may be advantageous to test other non-ionic detergents (Triton X100, Nonidet P40 or n-dodecyl-ßD-glucopyranoside) used commonly for fluorescence microscopy may create "bigger membrane holes" under milder conditions.
Intracellular cytokines staining
Incubation is done in permeabilization medium at 4°C for 30 min with an optimal dose of fluorochrome-conjuguated anti-cytokine antibody. Cell wash is performed in permeabilization buffer. Cells are thoroughly resuspensed prior to analysis of the samples by Flow Cytometry. Generally direct staining with antibody-fluorochrome conjugates give the best results, however, indirect staining of unconjugated antibodies with secondary (i.e., anti-Mouse Ig) fluorochrome labelled antibodies has been successful.
References
- Schmitz, J., Assenmacher, M. and A. Radbruch. (1993). Regulation of Th cell cytokine expression: functional dichotomy of APCs. Eur. J. Immunol. 23: 191.
- Jung, T., Schauer U., Heusser C., Neumann C. And C. Rieger (1993). Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159:197.
- Assenmacher, M., Schmitz, J. and A. Radbruch. (1994). Flow cytometric determination of cytokines in activated murine Th lymphocytes. Expression of IL-10 in IFN-gamma and in IL-4 expressing cells. Eur. J. Immunol. 24:1097.
- Prussin, C. & D.D. Metcalfe (1995). Detection of intracytoplasmic cytokines using flow cytometry and directly conjugated anticytokine antibodies. J. Immunol. Methods 188:117.