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The identification
of anti-cytokine monoclonal antibodies useful for staining intracellular
cytokines in cell suspension has provided the tools for multiparameter
flow cytometric analyses of individual cytokine-producing cells. The protocol
can be used to identify the phenotype and frequency of cell types defined
by membrane antigens and intracellular cytokines.
GENERAL PROCEDURE
Culture and stimulation of cells
Brefeldin A must be included in
cell culture systems. This agent block intracellular transport processes
resulting in the accumulation of most cytokine proteins in the Golgi complex,
enhancing the ability to detect cytokine-producing cells.
Brefeldin A (1-5 m
g/ml) have been included in in vitro cultures for 2 -4 hours prior to cell
harvest.
Stain cell surface antigens
Stain 106 cells in
100 m
l of PBS, 1% heat-inactivated FCS or BSA, pH 7.4 (staining buffer) with
appropriate concentrations of fluorochrome-conjugated monoclonal antibody
specific for a cell surface antigen.
Wash cells 2 times with staining
buffer and pellet by centrifugation (250 x g)
Fix cells
Thoroughly resuspend and fix cells
with 100 m
l of 4% paraformaldehyde, pH 7.4 for 20 min. at 4°C.
Wash cells 2 times with staining
buffer and pellet by centrifugation (250 x g)
Permeabilize Cells
Resuspend fixed cells in 100 m
l of staining buffer + 0.1% saponin containing a optimal concentration
of fluorochrome-conjugated anti-cytokine antibody incubate at 4°C for
30 minutes in the dark..
Wash cells 2 times with staining
buffer and pellet by centrifugation (250 x g)
References
Prussin, C., Metcalfe, D. Detection
of intracytoplasmatic cytokine using flow cytometry and directly conjugated
anticytokine antibodies. J.Immunol.Meth., 188: 117-128, 1995.
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone:(765) 494-0757; FAX (765) 494-0517; Web http://www.cyto.purdue.edu, EMAIL cdrom3@flowcyt.cyto.purdue.edu