Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is ≤0.25 ?g per million cells in 100 ?L?volume. It is recommended that the reagent be titrated for optimal performance for each application.

* Alexa Fluor? 488 has a maximum emission of 519 nm when it is excited at 488 nm.

7A6 antibody detects endogenous human NFATC1 in Western blot with an approximate molecular weight of 100 - 120 kD (different isoforms) (Cat. No. 649602). The optimal concentration should be determined by titration for each individual assay of interest.

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Clone 7A6 Intracellular Staining Procedure:

1. Prepare the fix/perm solution, consisting of 1% paraformaldehyde in 70% ethanol. Chill for at least one hour at -20°C.
2. Suspend cells in cell staining buffer and perform surface stain if necessary.
3. Centrifuge for 5 minutes at 1500 RPM and discard supernatant.
4. Resuspend cells in 500 ?L of fix/perm solution for up to 2 x 10^6 cells and gently vortex.
5. Incubate cells on ice for 30-60 minutes in the dark.
6. Add 2 mL PBS and centrifuge cells for 1500 RPM for 5 minutes.
7. Wash cells 1X with 2 mL cell staining buffer, centrifuge at 1500 RPM for 5 minutes, discard supernatant.
8. Adjust cell concentration to about 1 x 10^7 cells/mL, add 100 ?L (~ 1 x 10^6 cells) to a FACS tube that already contains the staining antibody and mix gently.
9. Incubate 15 minutes at room temperature.
10. Wash 2X with cell staining buffer as in step 6.
11. Resuspend cells in cell staining buffer and analyze.

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Cytoplasm, Nucleus. NFATc1 is cytoplasmic in the phosphorylated form and nuclear after calcineurin-mediated dephosphorylation controlled activation.

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