Protocols

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BD Phosflow Protocols for Human PBMCs

Protocol I (Detergent Method)

Fix Buffer used: BD Cytofix Buffer (Cat. No. 554655) or BD Phosflow Fix Buffer I (Cat. No. 557870)
Perm Buffer used: BD Phosflow Perm/Wash Buffer I (Cat. No. 557885)

1. Prepare PBMCs from normal or patient blood.
2. Pre-warm the BD Cytofix Buffer or BD Phosflow Fix Buffer I in a 37°C water bath for 5-10 minutes before use.
3. (Optional) Culture PBMCs in RPMI with 5% human serum at 37°C CO₂ incubator for 2 hrs.
   (This step may help to improve the signal for some phospho markers.)
4. Treat the cells with appropriate stimulators.
   (Note: Methods of activation varies with each phosphorylated cell signaling molecule; it should be determined by the researcher. Visit www.bdphosflow.com for some stimulators that have been tested.)
5. Fix the cells immediately in order to maintain their phosphorylation state. Rather than spinning down the cells, we recommend fixing the cells by adding an equal volume of pre-warmed BD Cytofix& Buffer or BD Phosflow Fix Buffer I to the cell suspension. Mix by inverting the tubes or brief vortexing.
6. Incubate the cells at 37°C for 10-15 minutes.
7. Optional: At this step, spin down the fixed cells, resuspend in the freezing buffer (PBS or media with 10% DMSO) and keep at -80°C up to 7 days for use at a later time. The frozen cells should be thawed at 37°C and washed immediately before proceeding with the following procedure.
8. Pellet the cells by centrifugation (300 x g) for 5-10 minutes and remove the supernatant.
9. Permeabilize the cells by adding BD Phosflow Perm/Wash Buffer I (1-10 x 10⁶ cells/ml, minimum 1 ml) and incubating for 10 minutes at room temperature.
   (This step can be omitted if using BD Phosflow Fix Buffer I for fixing the cells).
10. Wash the cells twice (once if using step 9) with BD Phosflow Perm/Wash Buffer I. Centrifuge at 300 x g for 5-10 minutes and remove supernatant.
11. Resuspend the cells in BD Phosflow Perm/Wash Buffer I at 1 x 10⁷ cells/ml.
12. Aliquot optimal concentration of fluorochrome-conjugated antibodies to each tube and add 100 µl (1 x 10⁶) of cells.
13. Incubate at room temperature for 30 minutes in the dark.
14. Wash once with 2 ml of BD Phosflow Perm/Wash Buffer I. Centrifuge at 300 x g for 5-10 minutes and remove supernatant. Resuspend in 500 µl of BD Pharmingen Stain Buffer (Cat. No. 554656) prior to flow cytometric analysis.
    

Protocol II and III (Mild or Harsh Alcohol Method)

Fix Buffer used: BD Cytofix Buffer (Cat. No. 554655)
Perm Buffer used: Depending on the surface markers and phospho-specific antibodies used, either BD Phosflow Perm Buffer II (Cat. No.558052) or III (Cat. No.558050) is recommended.

Please refer to our on-line Phospho-antibody and CD marker reference chart for details.

1. Prepare PBMCs from normal or patient blood.
2. Pre-warm the BD Cytofix Buffer in a 37°C water bath for 5-10 minutes before use.
3. (Optional) Culture PBMCs in RPMI with 5% human serum at 37°C CO2 incubator for 2 hrs.
   (This step may help to improve the signal for some phospho markers).
4. Treat the cells with appropriate stimulators.
   (Note: Methods of activation varies with each phosphorylated cell signaling molecule; it should be determined by the researcher. Visit www.bdphosflow.com for some stimulators that have been tested.)
5. Fix the cells immediately in order to maintain their phosphorylation state. Rather than spinning down the cells, we recommend fixing the cells by adding an equal volume of pre-warmed BD Cytofix Buffer to the cell suspension. Mix by inverting the tubes or brief vortexing.
6. Incubate the cells at 37°C for 10-15 minutes.
7. Optional: At this step, spin down the fixed cells, resuspend in the freezing buffer (PBS or media with 10%DMSO) and keep at -80°C up to 7 days for use at a later time. The frozen cells should be thawed at 37°C and washed immediately before proceeding with the following procedure.
8. Pellet the cells by centrifugation (300 x g) for 5-10 minutes and remove the supernatant.
9. Vortex or mix to loosen the pellet. Permeabilize the cells by adding BD Phosflow Perm Buffer II or III (1-10 x 10⁶ cells/ml, minimum 1 ml) (Cat. No.558052) and incubating for 30 minutes on ice.
   (Note: longer incubation times in this permeabilization buffer may decrease the signal intensity of surface marker staining. Perm Buffer II and III can be stored at room temperature or -20°C, but need to be at -20°C over night before use if stored at room temperature)
10. Wash cells twice with BD Pharmingen Stain Buffer (Cat. No. 554656). Centrifuge at 300 x g for 5-10 minutes and remove supernatant.
11. Resuspend cells in BD Pharmingen Stain Buffer at 1 x 10⁷ cells/ml.
12. Aliquot optimal concentration of fluorochrome-conjugated antibodies to each tube and add 100 µl (1 x 10⁶) of cells.
13. Incubate at room temperature for 30 minutes in the dark.
14. Wash once with 2 ml of BD Pharmingen Stain Buffer. Centrifuge at 300 x g for 5-10 minutes and remove supernatant. Resuspend in 500 µl of the same buffer prior to flow cytometric analysis.