Protocols

Printable View

BD Phosflow Protocols for Human Whole Blood Samples

Protocol I (Detergent Method)

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

1. Collect normal or patient 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 BD Phosflow™ Lyse/Fix Buffer to 1x with distilled water.
   (Example: 1 ml of 5x Lyse/Fix Buffer + 4 ml of DI water)
3. Pre-warm the 1x BD Phosflow™ Lyse/Fix Buffer in a 37°C water bath for 5-10 minutes before use.
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 cells immediately in order to maintain their phosphorylation state. Rather than spinning down the cells, we recommend fixing the cells by mixing one volume of blood with 20 volumes of the pre–warmed 1x BD Phosflow™ Lyse/Fix Buffer. Mix by inverting the tubes or brief vortexing.
6. Incubate the cells at 37°C for 10–15 minutes.
7. Pellet the cells by centrifugation (300 x g) for 5–10 minutes and remove the supernatant.
8. 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.
9. Pellet by centrifugation (300 x g) for 5–10 minutes and remove supernatant.
   
10. Wash cell once with BD Phosflow™ Perm/Wash Buffer I. Centrifuge at 300 x g for 5–10 minutes and remove supernatant.
11. Resuspend cells derived from 100–300 µl of blood in 100 µl of BD Phosflow™ Perm/Wash Buffer I.
12. Aliquot optimal concentration of fluorochrome–conjugated antibodies to each tube and add 100 µl 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 Phosflow™ Lyse/Fix Buffer (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.

1. Collect normal or patient 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 BD Phosflow™ Lyse/Fix Buffer to 1x with distilled water.
   (Example: 1 ml of 5x Lyse/Fix Buffer + 4 ml of DI water)
3. Pre–warm the 1x BD Phosflow™ Lyse/Fix Buffer in a 37°C water bath for 5–10 minutes before use.
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 cells immediately in order to maintain their phosphorylation state. Rather than spinning down the cells, we recommend fixing the cells by mixing one volume of blood with 20 volumes of the pre–warmed 1x BD Phosflow™ Lyse/Fix Buffer. Mix by inverting the tubes or brief vortexing.
6. Incubate the cells at 37°C for 10–15 minutes.
7. Pellet the cells by centrifugation (300 x g) for 5–10 minutes and remove the supernatant.
8. Wash cells once with PBS, and pellet by centrifugation (300 x g) for 5–10 minutes and remove 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) 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 derived from 100–300 µl of blood in 100 µl of BD Pharmingen™ Stain Buffer.
12. Aliquot optimal concentration of fluorochrome–conjugated antibodies to each tube and add 100µl 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.