Before intracellular staining of cytokines and other cytoplasmic and/or nuclear antigens, use a combination of membrane rupture buffer (10X ) and intracellular (IC) Fixation Buffer (Cat. No. 00-8222) or Foxp3 Transcription Factor Fix/Permeabilize Concentrate and Diluent (Cat. No. 00-5521) to fix and permeabilize cells. These solutions are specially formulated to reduce nonspecific staining of fluorochrome-labeled antibodies and improve fluorescence signal-to-noise ratio.

In the intracellular staining protocol, fixation is performed first, followed by membrane rupture. Wash steps, antibody dilutions, and incubations performed after membrane rupture should use membrane rupture buffer. A final wash step and cell resuspension should be completed using flow staining buffer before running samples.

Reported Applications
Intracellular staining followed by flow cytometric analysis