BD™ Phosflow Protocols for Mouse Splenocytes or Thymocytes

Protocol I (Detergent Method)

Fix Buffer used: BD™ Phosflow Lyse/Fix Buffer I (Cat. No. 558049)
Perm Buffer used: BD™ Phosflow Perm/Wash Buffer I (Cat. No. 557885)

1. Suspend thymocytes or splenocytes from whole thymus or spleen cells.
2. Treat cells with appropriate stimulators. Note: Methods of activation vary and should be determined by researcher.
3. Fix the cells immediately in order to maintain phosphorylation state. Rather than spinning down the cells, we recommend fixing cells by adding 20 volumes of pre-warmed 1x BD™ Phosflow Lyse/Fix Buffer to the cell suspension.
4. Incubate the cells at 37°C for 10 minutes. Pellet by centrifugation (300 x g) for 5-10 min and remove supernatant.
5. Vortex or mix to disrupt the pellet. Permeabilize the cells by adding 1 ml of BD™ Phosflow Perm/Wash Buffer I for 1-10 x 10⁶ cells and incubate for 10 min at room temperature.
6. Pellet the cells by centrifugation (300 x g) for 5-10 min and remove the supernatant.
7. Wash the cells once with BD Phosflow Perm/Wash Buffer I. Centrifuge at 300 x g for 5-10 min and remove the supernatant.
8. Resuspend the cells in BD Phosflow Perm/Wash Buffer I at 1 x 10 ⁷ cells/ml.
9. 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 min.
10. Aliquot 100 µl of the cell suspension (1 x 10⁶ cells) to each tube and add the recommended volumes of BD™ Phosflow antibodies. NOTE: Phospho and cell surface antigens can be stained simultaneously.
11. Incubate the cells at room temperature for 30 min in the dark.
12. Wash cells once with 2 ml of BD Phosflow 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)

Fix Buffer used: BD™ Phosflow Lyse/Fix Buffer I (Cat. No. 558049)
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:
Phosflow Application Handbook

1. Prepare single cell suspensions from mouse thymus or spleen in PBS with 5% FBS (no azide). Wash once with PBS - 5% FBS. Keep the cells at 4°C before stimulation.
2. Treat cells with appropriate stimulators. Note: Methods of activation vary and should be determined by researcher.
3. Fix the cells immediately in order to maintain phosphorylation state by mixing one volume of single cell suspension with 20 volumes of pre-warmed 1x BD™ Phosflow Lyse/Fix Buffer to the cell suspension.
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. Look in the Phosflow Application Handbook for some stimulators that have been tested. )

5. Incubate the cells at 37°C for 10 min. Pellet the cells by centrifugation (300 x g) for 5-10 min and remove the supernatant.
6. Vortex or mix to disrupt the pellet. Permeabilize the cells by adding 1 ml BD™ Phosflow Perm Buffer II or III (1-10 x 10⁶ cells/ml) (Cat. No.558052) and incubating for 30 min on ice.
7. Wash cells twice with BD Pharmingen™ Stain Buffer (Cat. No. 554656). Centrifuge at 300 x g for 5 min and remove supernatant.
8. Resuspend cells in BD Pharmingen Stain Buffer at 1 x 10⁷ cells/ml.
9. 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 min.
10. Aliquot optimal concentration of fluorochrome-conjugated antibodies to each tube and add 100 µl (1 x 10 ⁶ ) of cells. NOTE: Phospho and cell surface antigens can be stained simultaneously, however if there is difficulty maintaing cell surface staining, surface staining can be done immediately after fixation. (See Phosflow FAQ for more information)
11. Incubate at room temperature for 30 min in the dark.
12. Wash once with 2 ml of BD Pharmingen Stain Buffer and resuspend in the same buffer prior to flow cytometric analysis.