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Protocol: Intracellular Antibody Staining for Flow Cytometry

Intracellular Staining for Flow Cytometry

There are many methods for fixation and permeabilization of cells for flow cytometry. Protocols may need to be optimized for different cell types, targets, or applications. This is our basic protocol for intracellular staining of cells in suspension with antibodies for flow cytometry using our Flow Cytometry Fixation/Permeabilization Kit. For staining of extracellular cell surface antigens, see our Protocol: Cell Surface Antibody Staining for Flow Cytometry.

Materials required:

Workflow overview:

  1. Aliquot cells to flow tubes
  2. Fixation (20 min.)
  3. Wash and centrifuge (5 min.)
  4. Permeabilization/primary antibody incubation (30 min.)
  5. Wash and centrifuge (5 min.) 2x
  6. Secondary antibody incubation (not required for labeled primary) (30 min.)
  7. Wash and centrifuge (5 min.) 2x (optional stopping point)
  8. Analyze by flow cytometry

Procedure:

  1. Detach adherent cells from substrate by trypsinization or with a commercial non-enzymatic cell lift solution.
  2. Optional: To exclude dead cells from analysis, resuspend cells in PBS and stain with a fixable dead cell dye, such as our Live-or-Dye™ Fixable Viability Stains according to the product protocol.
  3. Optional: Perform antibody staining for cell surface markers (see Cell Surface Antibody Staining for Flow Cytometry).
  4. Adjust cell density to 107 cells per mL in PBS.
  5. Aliquot 100 uL cell suspension to each 12 x 75 mm polypropylene flow cytometry tubes for a total of 106 cells per tube.
  6. Add 100 uL fixation buffer to each tube and mix by gentle vortexing. Incubate at room temperature for 20 min.
    Note: If using directly labeled primary antibodies, protect tubes from light.
  7. Add 1 mL PBS to each tube and pellet cells by centrifugation for 5 min. at 350 x g.
  8. Pour off the wash buffer from the tubes into a waste container.
  9. Add 100 uL permeabilization buffer to each tube and mix by gentle vortexing.
  10. Add primary antibodies to the tubes and vortex gently to mix. Incubate at room temperature for 30 min.
    Note: Primary antibody concentration must be optimized for different applications, but 0.5-1 ug antibody per tube is a common starting concentration.
    Note: If using directly conjugated fluorescent primary antibodies, protect samples from light.
  11. Wash by adding 1 mL flow buffer to each tube. Pellet cells by centrifugation for 5 min. at 350 x g.
  12. Pour off the buffer into a waste container and repeat step 11.
  13. If using directly labeled primary antibodies, proceed to step 17. If using secondary antibodies, continue with step 14.
  14. After pouring off wash buffer, resuspend cells in the residual buffer (~100 uL) by gentle vortexing.
  15. Add 1 ug secondary antibodies to each tube and vortex gently to mix. Incubate at room temperature, protected from light, for 30 min.
    Note: For biotinylated primary antibodies, Streptavidin conjugates can be used for detection, typically at 0.25 ug/tube.
  16. Wash cells twice in flow buffer (repeat step 8-9).
  17. After pouring off wash buffer, add 500 uL of flow buffer per tube.
  18. Analyze by flow cytometry in the correct channel for your conjugate. Mix by gentle vortexing before loading each sample on cytometer.
    Note: Cells can be stored at 4°C, protected from light, for several days before analysis.