BD™ Phosflow Protocol for Human Whole Blood Samples

Procedural Notes

• 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).

Procedure

1. Collect normal or donor blood in the presence of anticoagulant. Depending on how many cells need to be collected, each staining tube will need 100–300 μL of blood.
2. Dilute 5x Lyse/Fix Buffer to 1x with distilled water (Example: 1 mL of 5x Lyse/Fix Buffer + 4 mL of distilled water).
3. Warm the 1x Lyse/Fix Buffer in a 37°C water bath for 5–10 minutes before use.
4. Treat the cells with appropriate stimulators.
5. Fix the cells immediately to maintain their phosphorylation state. Fix by mixing one volume of blood with 20-fold excess volume of warmed 1x Lyse/Fix Buffer. Mix by inverting the tubes or by briefly vortexing.
6. Incubate the cells at 37°C for 10–15 minutes.
7. Centrifuge the cells at 300g for 5–10 minutes and remove the supernatant.
8. 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.
9. Centrifuge the cells at 300g for 5–10 minutes and remove the supernatant.
10. Wash cells once with Perm/Wash Buffer I. Centrifuge at 300g for 5–10 minutes and remove the supernatant.
11. Resuspend cells derived from 100–300 μL of blood in 100 μL of Perm/Wash Buffer I.
12. Aliquot an optimal concentration of fluorochrome-conjugated antibodies to each tube and add 100 μL of cells. (Phospho and cell surface antigens can be stained simultaneously.)
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

• 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).
• Phospho and cell surface antignens can be stained simultaneously. However, if there is difficulty maintaining cell surface staining, it can be done immediately after fixation (step 5). (See Phosflow FAQ for more information.)
• Longer incubation times (step 9) 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. Collect normal or donor blood in the presence of anticoagulant. Depending on how many cells need to be collected, each staining tube will need 100-300 μL of blood.
2. Dilute 5x Lyse/Fix Buffer to 1x with distilled water (Example: 1 mL of 5x Lyse/Fix Buffer + 4 mL of distilled water).
3. Warm the 1x Lyse/Fix Buffer in a 37°C water bath for 5–10 minutes before use.
4. Treat the cells with appropriate stimulators.
5. Fix the cells immediately to maintain their phosphorylation state. Fix by mixing one volume of blood with 20-fold excess volume of warmed 1xLyse/Fix Buffer. Mix by inverting the tubes or by briefly vortexing.
6. Incubate the cells at 37°C for 10–15 minutes.
7. Centrifuge the cells at 300g for 5–10 minutes and remove the supernatant.
8. Wash the cells once with PBS, 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 II or III (1–10 x 10 6 cells/mL, minimum 1mL) 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 derived from 100-300 μL of blood in 100 μL of Stain Buffer.
13. Aliquot an optimal concentration of fluorochrome-conjugated antibodies to each tube and add 100 μL 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 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, whic 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 of phosphorylated protein specific antibosy sta8ining 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 cekk 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 Lyse/Fix Buffer to 37°C prior to use.
2. Dilute Perm Buffer IV to 1x using 1x Phosphate Buffered Saline (PBS) prior to use.
3. Treat 100 µL of whole blood cells with the selected activator and/or inhibitor.
4. Add a 20-fold excess volume (eg, 2.0 mL) of warmed Lyse/Fix Buffer. Mix well and incubate the tubes in a 37°C water bath for 10 to 15 minutes.
5. Centrifuge the cells at 400g for 10 minutes in a tabletop centrifuge. Aspirate the supernatant.
6. Wash the fixed cells once with PBS. Centrifuge the cells at 400g and aspirate the supernatant.
7. Vortex the tubes to loosen the cells.
8. 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 1 mL of Perm Buffer IV for cell concentrations less than 5 million cells.
9. 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-conjugates 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.