BD™ Phosflow Protocol for Human PBMCs

Suggested Reagents

*BD Cytofix™ Fixation Buffer and BD™ Phosflow Fix Buffer I have the same formulation.

Procedural Notes

• Culturing peripheral blood mononuclear cells (PBMCs) (step 3) might help improve the signal for some phospho markers.
• PBMCs can be prepared and then stored for up to 7 days for use at a later time (see step 7).
• Instead of centrifugation, we recommend fixing the cells by adding an equal volume of warmed Fix Buffer to the cell suspension.

Procedure

1. Prepare PBMCs from donor blood.
2. Warm the Fix 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 in a CO 2 incubator for 2 hours.
4. Treat the cells with appropriate stimulators.
5. Fix the cells immediately to maintain their phosphorylation state. Mix by inverting the tubes or briefly vortexing.
6. Incubate the cells at 37°C for 10–15 minutes.
7. (Optional) Centrifuge the fixed cells, resuspend them in the PBS or culture media with 10% DMSO and keep at –80°C up to 7 days. Thaw the cells at 37°C and wash them immediately before proceeding with the next step.
8. Centrifuge at 300g for 5–10 minutes and remove the supernatant.
9. If you are using BD Phosflow Fix Buffer I, skip to step 10. If you are using BD Cytofix fixation buffer, permeabilize the cells by adding Perm/Wash Buffer I (1–10 x 10 6 cells/mL, minimum 1 mL) and incubating for 10 minutes at room temperature.
10. Wash the cells twice (once if you performed step 9) with Perm/Wash Buffer I. Centrifuge at 300g for 5–10 minutes and remove the supernatant.
11. Resuspend the cells in Perm/Wash Buffer I at 1 x 10 7 cells/mL.
12. Aliquot an optimal concentration of fluorochrome-conjugated antibodies to each tube and add 100 μL (1 x 10 6) of cells.
13. Incubate at room temperature for 30 minutes in the dark.
14. Wash once with 2 mL of Perm/Wash Buffer I. Centrifuge at 300g for 5–10 minutes and remove the supernatant.
15. Resuspend in 500 μL of Stain Buffer prior to flow cytometric analysis.

Protocol II and III (Mild or Harsh Alcohol Method)

Suggested Reagents

*Select either Perm Buffer II or III depending on the surface markers and phosphospecific antibodies used.

Procedural Notes

• Culturing peripheral blood mononuclear cells (PBMCs) (step 3) might help improve the signal for some phospho markers.
• Methods of activation vary for each phosphorylated cell signaling molecule. Select stimulators before beginning the protocol. For stimulators that have been tested, see Techniques for Phospho Protein Analysis (Phosflow Application Handbook)
• Instead of centrifugation, we recommend fixing the cells by adding an equal volume of warmed Fix Buffer to the cell suspension.
• PBMCs can be prepared and then stored for up to 7 days for use at a later time (see step 7).
• Longer incubation times (step 10) in the permeabilization buffer could 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 overnight before use if stored at room temperature; prolonged chilling of the Perm Buffer II and III are required before use.

Procedure

1. Prepare PBMCs from normal or donor 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 in a CO 2 incubator for 2 hours.
4. Treat the cells with appropriate stimulators.
5. Fix the cells immediately to maintain their phosphorylation state. Mix by inverting the tubes or briefly vortexing.
6. Incubate the cells at 37°C for 10–15 minutes.
7. (Optional) Centrifuge the fixed cells, resuspend them in the PBS or culture media with 10% DMSO and keep at –80°C up to 7 days. Thaw the cells at 37°C and wash them immediately before proceeding with the next step.
8. Centrifuge at 300g for 5–10 minutes and remove the supernatant.
9. Vortex or mix to loosen the pellet.
10. Permeabilize the cells by adding Perm Buffer (1-10 x 10 6 cells/mL, minimum 1 mL) and incubating for 30 minutes on ice.
11. Wash the cells twice with Stain Buffer. Centrifuge at 300g for 5–10 minutes and remove the supernatant.
12. Resuspend the cells in Stain Buffer at 1 x 10 7 cells/mL.
13. Aliquot an optimal concentration of fluorochrome-conjugated antibodies to each tube and add 100 μL (1 x 10 6) of cells.
14. Incubate at room temperature for 30 minutes in the dark.
15. Wash once with 2 mL of Stain Buffer. Centrifuge at 300g for 5–10 minutes and remove the supernatant.
16. Resuspend in 500 μL of the same buffer prior to flow cytometric analysis.

Protocol IV (Harsh Detergent Method)

Suggested Reagents

Procedural Notes

• Be sure to remove all the supernatant after each centrifugation step. High residual volumes of supernatant will dilute the Perm Buffer IV, which could result in poor intracellular staining profiles.
• A longer than recommended permeabilization time, or using a ratio of cell to buffer volume outside the recommended ratio, could result in poorer fluorescent surface marker or phosphorylated protein specific antibody staining and detection. For a listing of compatible cell surface markers for use with Perm Buffer IV, visit http://static.bdbiosciences.com/documents/antibodies_human_cellsurface_marker.pdf
• Permeabilization with Perm Buffer IV could result in decreased cell recovery, an increase in time of permeabilization with Perm Buffer IV will increase cell loss.
• If staining of cell surface markers after permeabilization with Perm Buffer IV does not work well (there is a dim fluorescent signal), you can stain cells with fluorescent antibodies before cellular fixation (before step 4) or between the fixation and permeabilization steps. (after step 7) see below.

Procedure

1. Warm Fix Buffer to 37°C prior to use.
2. Dilute Perm Buffer IV to 1.0x using 1x Phosphate Buffered Saline (PBS) prior to use.
3. Isolate peripheral blood mononuclear cells (PBMCs) by density gradient separation (eg, using a CPT tube) from whole blood.
4. Wash the cells well and treat them with the selected activator, inhibitor, or combination.
5. At the end of the cell treatment, immediately mix one volume of the warmed Fix Buffer with one volume of the suspended PBMCs. Mix well and incubate the tubes in a 37°C water bath for 10 to 15 minutes.
6. Centrifuge the cells at 400g for 10 minutes in a tabletop centrifuge. Aspirate the supernatant.
7. Wash the fixed cells once with PBS. Centrifuge the cells at 400g and aspirate the supernatant.
8. Vortex the tubes to loosen the cells.
9. Permeabilize the cells by slowly adding Perm Buffer IV drop by drop to the cells. Add approximately 10 mL of Perm Buffer IV per 5 to 10 million cells. Add a minimum of 1mL of Perm Buffer IV for cell concentration less than 5 million cells. Incubate at room temperature for 15 to 20 minutes.
10. Centrifuge at 400g for 10 minutes and aspirate the supernatant.
11. Wash twice by adding Stain Buffer (FBS) to the cells. Centrifuge at 400g for 10 minutes and aspirate the supernatant each time.
12. Resuspend the cells in Stain Buffer (FBS) at 10 million cells/mL and aliquot 100 μL to each sample tube.
13. Aliquot an optimal concentration of phosphospecific fluorochrome-conjugated antibody to each tube. Incubate at room temperature for 30 minutes in the dark.
14. Wash once with 2 mL of Stain Buffer. Centrifuge at 300g for 5–10 minutes and aspirate the supernatant.
15. Resuspend in 500 μL of the same buffer prior to flow cytometric analysis.