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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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