Intracellular Flow Cytometry Protocol

Flow Cytometry Protocol

 

Overview:

Flow cytometry is a powerful tool with applications in multiple disciplines such as immunology, virology, molecular biology, and cancer biology.

 

Flow cytometry rapidly analyzes single cells as they flow past lasers while suspended in a buffered salt-based solution. Each particle is analyzed for visible light scatter and fluorescence parameters. Visible light scatter is measured in two different directions, the forward direction (Forward Scatter or FSC), which can indicate the relative size of the cell, and the Side Scatter (SSC), which shows the granularity of the cell.

This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.

 

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay).

  2. Centrifuge cells and discard supernatant.

  3. Add 1mL PBS (1% FBS) and centrifuge cell suspension at 2000 g for 4 minutes at 4℃. Discard the supernatant.

  4. Repeat Step 3 twice.

  5. Resuspend the cell pellet in 200 µL of Fixation/Permeabilization buffer. Incubate for 30 minutes at room temperature. Protect from light.

  6. Add 400 µL of 1x Permeabilization Buffer and centrifuge cell suspension at 2000 g for 5 minutes at room temperature. Discard the supernatant.

  7. Repeat Step 6 once.

  8. Block with 2% normal goat serum in 1x Permeabilization Buffer. Incubate for 15 minutes at room temperature.

  9. Without washing, add the recommended amount of primary antibody for detection of intracellular antigen(s) to cells and incubate for 60 minutes at room temperature.

  10. Add 300 µL of 1x Permeabilization Buffer and centrifuge cell suspension at 2000 g for 5 minutes at room temperature. Discard the supernatant.

  11. Repeat Step 10 once.

  12. Add the recommended amount of secondary antibody to each tube. Incubate for 30 minutes at room temperature. Protect from light.

  13. Add 400 µL of 1x Permeabilization Buffer and centrifuge cell suspension at 2000 g for 5 minutes at room temperature. Discard the supernatant.

  14. Repeat Step 13 once.

  15. Resuspend cells in 300 µL PBS and analyze on the flow cytometer.

 

Only for research applications, not for diagnostic or therapeutic use.

 

Suggested buffer compositions

Buffer

Composition

1x Phosphate Buffered Saline (PBS)

8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4.

Adjust the pH to 7.4 with HCl.

Fixation/Permeabilization Buffer

eBioscience™ Fixation/Permeabilization Concentrate(00-5123-43)

eBioscience™ Fixation/Permeabilization Diluent( 00-5223-56) Dilute 1 part Concentrate with 3 parts Diluent

Permeabilization Buffer

 

eBioscience™ Permeabilization Buffer (10X)(00-8333-56) Dilute tenfold in distilled water.

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