THREE TIPS IN METHODOLOGY
 
GIUSEPPE BASSO
UNIVERSITY OF TORINO
DEPARTMENT OF PEDIATRICS
 
Piazza Polonia, 94
Torino
tel. +39 11 3531204
fax +39 49 821.3510
E-mail: gbasso@child.pedi.unipd.it
 
IMMUNOFLUORESCENT STAINING OF INTRACELLULAR CYTOKINES FOR FLOW CYTOMETRIC ANALYSIS

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