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Cell Fixation/Permeabilization Kits for Intracellular Cytokine Analysis

Pharmingen offers three cell fixation/permeabilization kits to simplify the preparation of cells for intracellular staining of cytokines. All three kits enable one step fixation and permeabilization of cells.

1. BD Cytofix/Cytoperm™ Kit (Cat. No. 554714)

Kit components:

  • BD Cytofix/Cytoperm solution
  • BD Perm/Wash buffer (10X)
  • Detailed protocol with sample data

This kit enables the one step fixation and permeabilization of cells which is necessary prior to the staining of intracellular cytokines with fluorochrome-conjugated anti-cytokine antibodies. This kit provides two reagents: BD Cytofix/Cytoperm™ fixation/permeabilization solution and BD Perm/Wash™ staining buffer. After the cells are fixed and permeabilized with the BD Cytofix/Cytoperm solution, the BD Perm/Wash buffer is used to wash the cells and to dilute the anti-cytokine antibodies for staining. *It is important that the BD Perm/Wash buffer be used for dilution of anti-cytokine antibodies, rather than a standard staining buffer, in order to maintain cells in a permeabilized state for intracellular staining.

2. Fixation and Permeabilization Solution Kit with BD GolgiStop™ (Cat. No. 554715)

Kit components:

  • BD Cytofix/Cytoperm solution
  • BD Perm/Wash buffer
  • Detailed protocol with sample data
  • BD GolgiStop

In addition to the fixation/permeabilization and diluent/wash solutions included in the BD Cytofix/Cytoperm Kit, this kit includes BD GolgiStop, containing the protein transport inhibitor monensin. Addition of BD GolgiStop to cell activation cultures blocks intracellular transport processes, thereby resulting in the accumulation of most cytokine proteins in the endoplasmic reticulum and enhancing cytokine staining signal. Sufficient BD GolgiStop reagent is provided for treating 1 L of cultured cells.

WARNING: BD GolgiStop contains monensin, a chemical which is known to be toxic. Avoid contact with skin, eyes and mucous membranes.

NOTE: Because differential effects comparing monensin and brefeldin A have been observed on the detection of certain cytokines by intracellular cytokine staining, it is recommended that the researcher test both transport inhibitors in their experimental system to determine which one is optimal. Each inhibitor is also sold separately.

3. Fixation and Permeabilization Solution Kit with BD GolgiPlug™ (Cat. No. 555028)

Kit components:

  1. BD Cytofix/Cytoperm solution
  2. BD Perm/Wash buffer
  3. Detailed protocol with sample data
  4. BD GolgiPlug

In addition to the fixation/permeabilization and diluent/wash solutions included in the BD Cytofix/Cytoperm Kit, this kit includes BD GolgiPlug™, containing the protein transport inhibitor brefeldin A. Addition of BD GolgiPlug to cell activation cultures will block intracellular transport processes, thereby resulting in the accumulation of most cytokine proteins in the Golgi complex and enhancing cytokine staining signal. Sufficient BD GolgiPlug reagent is provided for treating 1 L of cultured cells.

WARNING: BD GolgiPlug contains Brefeldin A, a chemical which is known to be toxic. Avoid contact with skin, eyes and mucous membranes.

NOTE: Because differential effects comparing monensin and brefeldin A have been observed on the detection of certain cytokines by intracellular cytokine staining, it is recommended that the researcher test both transport inhibitors in their experimental system to determine which one is optimal. Each inhibitor is also sold separately.

WARNINGS: BD Cytofix/Cytoperm contains formaldehyde, a suspected carcinogen. Avoid contact with skin, eyes and mucous membranes. Avoid breathing fumes. The BD Perm/Wash solution contains sodium azide and saponin.

CAUTIONS:

  • Harmful by inhalation, in contact with skin, and if swallowed
  • Possible risks of irreversible effects
  • May cause sensitization by skin contact • In case of contact with eyes, rinse immediately with plenty of water and seek medical advice
  • In case of accident or if you feel unwell, seek medical advice immediately. (Show the label where possible)
  • Wear suitable protective clothing and gloves • Use only in well-ventilated areas.

References:

Carter, L. L., and S. L. Swain. 1997. Single cell analyses of cytokine production. Curr. Opin. Immunol. 9:177-182.

Parks, D. R., L. A. Herzenberg, and L. A. Herzenberg. 1989. Flow cytometry and fluorescence-activated cell sorting. In Fundamental Immunology, 2nd Edition. W. E. Paul, ed. Raven Press Ltd., New York, p. 781-802.

Jung, T., U. Schauer, C. Heusser, C. Neumann and C. Rieger. 1993. Detection of intracellular cytokines by flow cytometry. J. Immunol. Meth. 159:197-207.

Vikingson, A., K. Pederson and D. Muller. 1994. Enumeration of IFN-gproducing lymphocytes by flow cytometry and correlation with quantitative measurement of IFN-g. J. Immunol. Meth. 173:219-228.

Prussin, C. and D. Metcalfe. 1995. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J. Immunol. Meth. 188: 117-128.

Elson, L. H., T. B. Nutman, D. D. Metcalfe and C. Prussin. 1995. Flow cytometric analysis for cytokine production identifies Th1, Th2, and Th0 cells within the human CD4 + CD27 - lym-phocyte subpopulation. J. Immunol. 154:4294-4301.

Assenmacher, M., J. Schmitz and A. Radbruch. 1994. Flow cytometric determination of cytokines in activated murine T helper lymphocytes: Expression of interleukin-10 in interferon-g and in interleukin-4-expressing cells. Eur. J. Immunol. 24:1097-1101.

Picker, L. J., M. K. Singh, Z. Zdraveski, J. R. Treer, S. L. Waldrop, P. R. Bergstresser, and V. C. Maino. 1995. Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 86:1408-1419.

Sallusto, F., C. R. Mackay, and A. Lanzavecchia. 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005-2007.

Austrup, F., D. Vestweber, E. Borges, M. Löhning, R. Bräuer, U. Herz, H. Renz, R. Hallmann, A. Scheffold, A. Radbruch, and A. Hamann. 1997. P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues. Nature 385:81-83.

Sander, B., J. Andersson and U. Andersson. 1991. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol. Rev. 119:65-93.

Sander, B., I. Hoiden, U. Andersson, E. Moller, and J. Abrams. 1993. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. J. Immunol. Meth. 166:201-214.

Anderson, U. and J. Andersson. 1994. Immunolabelling of cytokine producing cells in tissues and suspension. In Cytokine Producing Cells, eds. D. Fradelizie and D. Emelie. INSERM, Paris. p. 32-49.

Ferrick, D. A., M. D. Schrenzel, T. Mulvania, B. Hsieh, W. G. Ferlin and H. Lepper. 1995. Differential production of interferon-gand interleukin-4 in response to Th1- and Th2-stimulating pathogens by gdT cells in vivo. Nature. 373:255-257.

Sornasse, T., P. V. Larenas, K. A. Davis, J. E. de Vries, and H. Yssel. 1996. Differentiation and stability of T helper 1 and 2 cells derived from naive human neonatal CD4 + T cells, analyzed at the single cell levels. J. Exp. Med. 184:473-483.

Andersson, S. and C. Coleclough. 1993. Regulation of CD4 and CD8 expression on mouse T cells. J. Immunol. 151:5123-5134.