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).
• Instead of centrifugation, we recommend fixing cells (step 3) by adding 20-fold excess volume of warmed 1x Lyse/Fix Buffer I to the cell suspension.

Procedure

1. Suspend thymocytes or splenocytes from whole thymus or spleen cells.
2. Treat the cells with appropriate stimulators.
3. Fix the cells immediately to maintain the phosphorylation state. (See Procedural Notes.)
4. Incubate the cells at 37°C for 10 minutes.
5. Centrifuge at 300g for 5 to 10 minutes and remove the supernatant.
6. Vortex or mix to disrupt the pellet.
7. Permeabilize the cells by adding 1 mL of Perm/Wash Buffer I for 1–10 x 10 6 cells and incubate for 10 minutes at room temperature.
8. Centrifuge at 300g for 5 to 10 minutes and remove the supernatant.
9. Wash the cells once with Perm/Wash Buffer I. Centrifuge at 300g for 5 to 10 minutes and remove the supernatant.
10. Resuspend the cells in Perm/Wash Buffer I at a the final cell count of 1 x 10 7 cells/mL.
11. Add 0.06 μg of BD™ Fc Block antibody (Cat. No. 553141 or 553142) for each 1 x 10 6 cells. Incubate on ice for 15 minutes.
12. Aliquot 100 μL of the cell suspension (1 x 10 6 cells) to each tube and add the recommended volumes of BD Phosflow antibodies. (Phospho and cell surface antigens can be stained simultaneously.)
13. Incubate the cells at room temperature for 30 minutes in the dark.
14. Wash cells once with 2 mL of Perm/Wash Buffer I and resuspend the cells in the same 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 antigens can be stained simultaneously. However, if there is difficulty maintaining cell surface staining, it can be done immediately after fixation (step 4). (See BD™ Phosflow FAQs for more information.)

Procedure

1. Prepare single-cell suspensions from mouse thymus or spleen in PBS with 5% FBS (no azide).
2. Wash once with PBS with 5% FBS. Keep the cells at 4°C before stimulation.
3. Treat the cells with appropriate stimulators.
4. Fix the cells immediately to maintain the phosphorylation state by adding one volume of single-cell suspension to 20 volumes of warmed 1x Lyse/Fix Buffer.
5. Incubate the cells at 37°C for 10 minutes. Centrifuge at 300g for 5 to 10 minutes and remove the supernatant.
6. Vortex or mix to disrupt the pellet.
7. Permeabilize the cells by adding 1 mL of Perm Buffer II or III for 1–10 x 10 6 cells and incubating for 30 minutes on ice.
8. Wash the cells twice with Stain Buffer. Centrifuge at 300 g for 5 minutes and remove the supernatant.
9. Resuspend the cells in Stain Buffer at a the final cell count of 1 x 10 7 cells/mL.
10. Add 0.06 μg of BD™ Fc Block antibody (Cat. No. 553141 or 553142) for each 1 x 106 cells. Incubate on ice for 15 minutes.
11. Aliquot an optimal concentration of fluorochrome-conjugated antibodies to each tube and add 100 μL (1 x 10 6) of cells.
12. Incubate at room temperature for 30 minutes in the dark.
13. Wash once with 2 mL of Stain Buffer and resuspend in 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.
• Permeabilization with Perm Buffer IV could result in decreased cell recovery, 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 9) see below.

Procedure

1. Warm Lyse/Fix Buffer I to 37°C.
2. Dilute Perm Buffer IV to 1.0x using 1x Phosphate Buffered Saline (PBS) prior to use.
3. Prepare single-cell suspensions from mouse thymus or spleen in PBS with 5% FBS (no azide).
4. Wash once with PBS with 5% FBS. Keep the cells at 4°C before stimulation.
5. Treat the cells (example: use 100 μl of cell suspension) with the selected activator and/or inhibitor for the selected period of time.
6. After treatment, add a 20-fold excess volume (eg, 2.0 mL) of warmed Lyse/Fix Buffer I. Mix well and incubate the tubes in a 37°C water bath for 10 to 15 minutes.
7. Centrifuge the cells at 400g for 10 minutes in a tabletop centrifuge. Aspirate the supernatant.
8. Wash the fixed cells once with PBS. Centrifuge at 400g and aspirate the supernatant.
9. Vortex the tubes to loosen the cells.
10. 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. For cell concentrations less than 5 million, add a minimum of 1 mL of Perm Buffer IV.
11. Incubate at room temperature for 15 to 20 minutes.
12. Centrifuge at 400g for 10 minutes and aspirate the supernatant.
13. Wash twice by adding Stain Buffer (FBS) to the cells. Centrifuge at 400g for 10 minutes and aspirate the supernatant each time.
14. Resuspend the cells in Stain Buffer (FBS) at 10 million cells/mL and aliquot 100 μL to each sample tube.
15. Aliquot an optimal concentration of phosphospecific, fluorochrome-conjugated antibody to each tube. Incubate at room temperature for 30 minutes in the dark.
16. Wash once with 2 mL of Stain Buffer prior to flow cytometric analysis.