Flow cytometry protocol

Flow Cytometry Protocol: Intracellular Staining Intracellular flow cytometry can be used to analyze a variety of intracellular molecules including cytokines, inflammatory mediators, transcription factors, and phosphoproteins. It can provide rich information concerning cellular function and signaling responses. Different from cell surface markers staining, intracellular antigens detection requires cell fixation and permeabilization before staining.

Typically, cells are fixed with formaldehyde to preserve the cellular morphology, and then permeabilized with detergent or alcohol. Such fixation/permeabilization treatment allows the antibodies against intracellular antigens across the plasma membrane to stain intracellularly, while maintaining the morphological characteristics of cells.

Notes: For staining of secreted proteins, such as cytokines, a protein transport inhibitor such as Brefeldin A or Monensin, should be added prior to fixation/permeabilization in order to trap the cytokines inside the cells and enable intracellular staining.

When surface and intracellular staining is to be performed in the same sample. It is advised that surface staining should be carried out first to avoid any potential effects of the intracellular staining protocol.

Reagents: PBS: Dissolve 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4 and 0.2 g KH2PO4 in 800 mL distilled water. Adjust the pH to 7.4 with HCl and final volume to 1 liter with additional distilled H2O. Cell staining buffer: Add 0.5% BSA and 0.05% Sodium Azide (NaN3) to PBS. Fixation buffer: 1% paraformaldehyde in PBS. Permeabilization buffer: 0.1% saponin in cell staining buffer. https://www.creative-diagnostics.com/flow-cytometry-protocol-intracellular-staining. htm