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

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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 Buffer to 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 Buffer to 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.  

 

General Tips & FAQ

• What are some improvements in the True-Nuclear™ Buffer compared to the Foxp3 Fix/Perm (Cat. No. 421403) and the Nuclear Factor Fixation and Permeabilization Buffer Sets (Cat. No.422601)?
  • Less background for nuclear staining (e.g.- Foxp3) staining, offering improved signal/noise ratio for better population distinction
  • Minimal effects on surface staining, especially those stained using tandem fluorophores such as APC/FIRE™ 750

• I am observing high background staining. What can I do to reduce it?
  • Longer fixation time – as noted in the fixation step, a longer fixation period can help ameliorate high background

• Is the True-Nuclear™ Buffer set recommended for intracellular cytokine staining?
  • Permeabilization conditions required for staining transcription factors using True-Nuclear™ (or Foxp3 Fix/Perm and Nuclear Factor Fixation and Permeabilization Buffer Sets) is much harsher than those generally recommended for staining cytokines (i.e.- cells become more porous). Therefore, cytokine leakage upon permeabilization and resulting signal loss is a potential concern. However, numerous users have reported successful staining for both nuclear and cytoplasmic targets simultaneously using the True-Nuclear™ buffer set. For staining only intracellular cytokines, we recommend using the Fixation (Cat. No. 420801) and Permeabilization Wash Buffers (Cat. No. 421002) and following our intracellular staining protocol, which can be found here.

• Any known nuclear targets not suited for detection using the True-Nuclear™ buffer?
  • Ki-67: You can find our recommended protocol for Ki-67 staining here