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)
