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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.
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1.
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BD Cytofix/Cytoperm™ Kit (Cat. No. 554714)
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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.
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2.
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Fixation and Permeabilization Solution
Kit with BD GolgiStop™ (Cat. No. 554715) |
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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.
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3.
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Fixation and Permeabilization Solution Kit with
BD GolgiPlug™ (Cat. No. 555028)
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Kit components:
• BD Cytofix/Cytoperm solution
• BD Perm/Wash buffer
• Detailed protocol with sample data
• 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.
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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:
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1.
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Carter, L. L., and S. L. Swain. 1997. Single cell analyses
of cytokine production. Curr. Opin. Immunol.9:177-182.
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2.
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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.
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3.
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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.
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4.
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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.
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5.
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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.
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6.
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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.
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7.
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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.
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8.
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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. Blood86:1408-1419.
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9.
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Sallusto, F., C. R. Mackay, and A. Lanzavecchia. 1997.
Selective expression of the eotaxin receptor CCR3 by human T helper 2
cells. Science277:2005-2007.
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10.
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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. Nature385:81-83.
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11.
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Sander, B., J. Andersson and U. Andersson. 1991.
Assessment of cytokines by immunofluorescence and the
paraformaldehyde-saponin procedure. Immunol. Rev. 119:65-93.
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12.
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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.
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13.
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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.
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14.
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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.
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15.
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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.
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16.
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Andersson, S. and C. Coleclough. 1993. Regulation of CD4
and CD8 expression on mouse T cells. J. Immunol. 151: 5123-5134.
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