True - Nuclear™ Transcription Factor Staining Protocol for 96 - Well , U - Bottom Plate
Perform cell surface staining as described in BioLegend's Cell Surface Immunofluorescence Staining Protocol .
After the last wash , discard the supernatant , and gently vortex the samples to dissociate the cell pellet .
Add 200 µL of the Transcription Factor 1X Fix solution to each well .
Gently pipette to ensure cells are fully resuspended .
Centrifuge the plate at 300 - 400 x g at room temperature for 5 minutes , discard the supernatant , and gently vortex to dissociate the cell pellet .
Add 200 µL of the Transcription Factor 1X Perm Buffer to each well . ( wash 1 / 3 )
Centrifuge the plate at 300 - 400 x g at room temperature for 5 minutes , discard the supernatant , and gently vortex to dissociate the cell pellet . ( wash 1 / 3 )
Add 200 µL of the Transcription Factor 1X Perm Buffer to each well . ( wash 2 / 3 )
Add the appropriate amount of fluorochrome conjugated antibody for detection of intracellular antigen ( s ) to each well and incubate in the dark at room temperature for at least 30 minutes .
Add 200 µL of the Transcription Factor 1X Perm Buffer to each well . ( wash 1 / 3 )
Centrifuge the plate at 300 - 400 x g at room temperature for 5 minutes , discard thesupernatant , and gently vortex to dissociate the cell pellet . ( wash 1 / 3 )
Resuspend in cells in appropriate volume of cell staining buffer and acquire samples on a flowcytometer .
Incubate at room temperature in the dark for 30 - 60 minutes .
Add 200 µL of the Transcription Factor 1X Perm Buffer to each well . ( wash 3 / 3 )
Centrifuge the plate at 300 - 400 x g at room temperature for 5 minutes , discard the supernatant , and gently vortex to dissociate the cell pellet . ( wash 2 / 3 )
Centrifuge the plate at 300 - 400 x g at room temperature for 5 minutes , discard the supernatant , and gently vortex to dissociate the cell pellet . ( wash 3 / 3 )
Add 200 µL of the Transcription Factor 1X Perm Buffer to each well . ( wash 2 / 3 )
Centrifuge the plate at 300 - 400 x g at room temperature for 5 minutes , discard thesupernatant , and gently vortex to dissociate the cell pellet . ( wash 2 / 3 )
Add 200 µL of the Transcription Factor 1X Perm Buffer to each well . ( wash 3 / 3 )
Centrifuge the plate at 300 - 400 x g at room temperature for 5 minutes , discard thesupernatant , and gently vortex to dissociate the cell pellet . ( wash 3 / 3 )
