Flow cytometry can be used to analyze various intracellular molecules including phosphorylated signaling proteins and cytokines. Cytokines and other secreted molecules can be detected by flow cytometry in activated cells with the aid of secretion inhibitors, such as monensin or brefeldin A. These compounds prevent the export of newly synthesized proteins by disrupting the ER-Golgi transport machinery. For experimental treatments with stimulation periods of up to 4-6 hours, the secretion inhibitor can be present during the entire incubation period. If the stimulation period is longer than 4-6 hours, the secretion inhibitor should be added for only the last two hours of the incubation.
There are many variables that must be optimized for individual flow cytometry experiments such as antibody incubation time and temperature. Furthermore, to stain intracellular molecules, the cells need to be fixed in suspension and then permeabilized before the detection antibody is added. This fixation/permeabilization treatment allows the antibody to pass through the plasma membrane into the cell interior, while maintaining the morphological characteristics used to sort the cells. Commonly used detergents include saponin, Triton® X-100, or Tween® 20. The following protocol has been developed and optimized by R&D Systems Flow Cytometry Laboratory for the staining of intracellular molecules for flow cytometric analysis.
Note: When surface and intracellular staining is to be performed in the same sample, it is advisable that the surface staining be performed first, since fixation/permeabilization treatments might decrease the availability of surface antigens.
Please read the protocol in its entirety before starting.
- PBS (1X): 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4 or Hank’s Balanced Salt Solution (HBSS; 1X)
- Flow Cytometry Fixation Buffer (R&D Systems, Catalog # FC004, or an equivalent solution containing 1 - 4% paraformaldehyde)
- Flow Cytometry Permeabilization Buffer/Wash Buffer I (1X; R&D Systems, Catalog # FC005, or an equivalent solution containing a permeabilization agent such as saponin or Triton X-100)
- (Optional, see Note in step 3) Flow Cytometry Fixation/Permeabilization Buffer I (1X; Catalog # FC007)
- Detection Antibodies
- Isotype Control Antibodies
- FACS™ Tubes (5 mL round-bottom polystyrene tubes)
- Pipette Tips and Pipettes
- Harvest the cells and wash 2 times by adding 2 mL of PBS (or HBSS), centrifuging at 300 x g for 5 minutes, and then decanting buffer from pelleted cells.
Note: Staining of surface antigens may be done at this point.
- Aliquot up to 1 x 106 cells/100 μL into FACS tubes. Add 0.5 mL of cold Flow Cytometry Fixation Buffer and vortex. Incubate at room temperature for 10 minutes. Vortex the cells intermittently in order to maintain a single cell suspension.
- Centrifuge cells and decant the Fixation Buffer. Wash the cells 2 times with PBS (or HBSS) as in step 1. Resuspend the cell pellet in 100 – 200 μL of Flow Cytometry Permeabilization/Wash Buffer I.
Note: Depending on the specific antibody and cell sample being used, the fixation and permeabilization steps can be performed simultaneously using Flow Cytometry Fixation/Permeabilization Buffer I.
- Add 10 μL of conjugated antibody (or a previously titrated amount) and vortex. Incubate cells for 30 minutes at room temperature in the dark.
Note: Because saponin-mediated cell permeabilization is a reversible process, it is important to keep the cells in the presence of Permeabilization Buffer I during intracellular staining.
- Wash cells 2 times with Flow Cytometry Permeabilization/Wash Buffer I as in step 3.
Note: If an unconjugated primary antibody was used, incubation with an appropriate secondary antibody should occur now. Dilute the secondary antibody in Flow Cytometry Permeabilization/Wash Buffer I, starting with the concentration suggested in the product datasheet. Incubate for 20-30 minutes in the dark and wash as in step 3.
- Resuspend the cells in 200 – 400 μL PBS (or HBSS) buffer for flow cytometric analysis.
Note: For a negative control, a separate set of cells should be stained with an isotype control antibody.
||CD220 ligation results in a dose-dependent increase of phosphorylated ERK1/ERK2. Mouse CD3+ T cells were stimulated with an immobilized Hamster Anti-Mouse CD3 epsilon Monoclonal Antibody (100 ng/mL, Catalog # MAB484) and the indicated concentrations of Recombinant Mouse CD229-His (Catalog # 2555-CD) at 37 °C, 5% CO2 for 1 hour. Cells were then washed, immediately fixed, and washed again. The cells were permeabilized using Flow Cytometry Fixation & Permeabilization Buffer (Catalog # FC007). After washing, the cells were blocked with a Rat Anti-Mouse Fc gamma RII/III (CD32/CD16) Monoclonal Antibody (Catalog # MAB1460) for 20 minutes before the addition of a Fluorescein-conjugated Rabbit Anti-Human/Mouse/Rat Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) Affinity-purified Polyclonal Antibody (Catalog # IC1018F) or a Fluorescein-conjugated Rabbit IgG Isotype Control (Catalog # IC105F) for an additional 45 minutes at room temperature in the dark.
FACS is a trademark of Becton Dickinson and Company.
Triton is a registered trademark of The Dow Chemical Company.
Tween is a registered trademark of Uniqema Americas LLC.