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INTRACELLULAR STAINING OF MOUSE CYTOKINES
REAGENTS
rat anti mouse cytokine mAbs
anti IL2 - S4B6 anti IL3 - 43D11 + 8F8 anti
IL4 - 11B11
anti IL10 - JES52A5 anti IFN-gamma - R46A2 (or AN18.17.24)
goat anti rat IgG-FITC H+L (x mouse) (Southern Biotechnology Associates)
buffer: - PBS and PBS/BSA/NaN3
- PBS/BSA/NaN3 with 0,5% Saponin (from Saponaria, Sigma)
(saponin-buffer)
fixative: - 4% formaldehyde (Merck) in PBS
PROCEDURE:
FIXATION
- wash cells once with PBS
- resuspend cells in PBS at 2*10^6/ml
- add 1Vol 4% formaldehyde/PBS
20' at RT
- wash 2x with PBS
- resuspend in PBS/BSA/NaN3 at 1-2*10^6 cells/ml and store at 4¡C in the dark
until
staining
INTRACELLULAR STAINING
- use about 1*10^6 cells for each 1.5ml test tube sample; spin down 10' at
300g
- incubate pellet with 30µl anti cytokine mab in saponin-buffer 15' at RT
- wash 2x with 1ml saponin-buffer
- incubate pellet with 30µl goat anti rat IgG-FITC in saponin-buffer 20'
at RT
- wash 1x with 1ml saponin-buffer
SURFACE STAINING
- wash 1x with PBS/BSA/NaN3 (without saponin!)
- incubate pellet with 30µl PE conjugated mAb in PBS/BSA/NaN3 10' at RT
- wash 1x with PBS/BSA/NaN3
---> flow cytometry/fluorescence microscopy
The same protocol can be used for human cytokines.
REFERENCES
Schmitz, J., Assenmacher, M. and A. Radbruch. 1993. Regulation of Th cell
cytokine expression: functional dichotomy of APCs. Eur. J. Immunol. 23: 191
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
Hope it works,
Alexander Horst
AlexanderH@MiltenyiBiotec.de
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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