True-Nuclear™ Transcription Factor Staining Protocol for 5mL Tubes

 

Reagent List

  • True-Nuclear™ Transcription Factor Buffer Set (Cat. No.424401)
  • Cell Staining Buffer (Cat. No. 420201)

 

Protocol Steps


  1. Perform cell surface staining as described in BioLegend's Cell Surface Immunofluorescence Staining Protocol.

  2. Add 1mL of the True-Nuclear™ 1X Fix Concentrate to each tube, vortex and incubate at room temperature in the dark for 45-60 minutes.

    If necessary, the protocol can be suspended at this point. After discarding supernatant, re-suspend cells in CytoLast™ Buffer (Cat. No. 422501) or equivalent. Samples can be stored at 4°C for 12-18 hours, protected from light and plastic-wrapped to protect buffer evaporation.

  3. Without washing, add 2mL of the True-Nuclear™ 1X Perm Bufferto each tube.

  4. Centrifuge tubes at 300-400xg at room temperature for 5 minutes, and discard the supernatant.

  5. Add 2mL of the True-Nuclear™ 1X Perm Buffer to each tube.

  6. Centrifuge tubes at 300-400xg at room temperature for 5 minutes, and discard the supernatant.

  7. Resuspend the cell pellet in 100µL of the True-Nuclear™ 1X Perm Buffer.

  8. Add the appropriate amount of fluorochrome conjugated antibody diluted in True-Nuclear™ 1X Perm Buffer for detection of intracellular antigen(s) to each well and incubate in the dark at room temperature for at least 30 minutes.

  9. Add 2mL of the True-Nuclear™ 1X Perm Bufferto each tube.

  10. Centrifuge tubes at 300-400 x g at room temperature for 5 minutes, and discard the supernatant.

  11. Add 2mL of cell staining buffer (Cat. No. 420201).

  12. Centrifuge tubes at 300-400xg at room temperature for 5 minutes, and discard the supernatant.

  13. Resuspend in 0.5mL cell staining buffer then acquire the tubes on a flow cytometer.  

 

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