1. Warm BD Phosflow™ Fix Buffer I (Cat. No. 557870) to 37 °C prior to use.
2. Chill BD Phosflow™ Perm Buffer II to -20 °C prior to use.
3. Collect PBMCs after Ficoll-Paque separation and treat with any appropriate cell activators as applicable.
4. At the end of the treatment, immediately mix one volume of the warmed BD Phosflow™ Fix Buffer I with one volume of the PBMC suspension. Mix well and incubate the tubes in a 37 °C water bath for 10 min. Spin down the cells at 250 x g for 10 min in a table-top centrifuge and then aspirate the supernatant.
5. Wash the cells once with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656). Alternatively, 1X Washing/Staining Solution (1 x PBS, 1% FCS, and 0.09% sodium azide) may be used. Centrifuge to pellet the cells and remove the supernatant.
6. Vortex to loosen the cells. Permeabilize the cells by slowly adding cold BD Phosflow™ Perm Buffer II while vortexing. Incubate on ice for 30 min and then spin down the cells.
7. Wash the cells twice with 5-10 mL of BD Pharmingen™ Stain Buffer (FBS). Centrifuge at 250 x g for 10 minutes and remove the supernatant by aspiration.
8. Resuspend cells in BD Pharmingen™ Stain Buffer (FBS) at 1x 10^7/ml and aliquot 100 µl to each flow tube to continue with antibody staining and flow cytometric analysis.
Danger: BD Phosflow™ Perm Buffer II contains 64.86% methanol (w/w).
Highly flammable liquid and vapor.
Toxic if swallowed, in contact with skin or if inhaled.
Causes damage to organs.
Keep away from heat/sparks/open flames/hot surfaces/ignition sources. - No smoking.
Keep container tightly closed. Ground and bond container and receiving equipment. Use explosion-proof [electrical/ventilating/lighting/] equipment.
Use non-sparking tools. Take action to prevent static discharges.
Wear protective clothing/eye protection/gloves.
Do not breathe mist/vapours/spray.
Use only outdoors or in a well-ventilated area. Wash thoroughly after handling. Do not eat, drink or smoke when using this product.
IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin with water/shower. Take off immediately all contaminated clothing. Wash contaminated clothing before reuse.
IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing.
IF SWALLOWED: Immediately call a POISON CENTER/doctor. Rinse mouth. Call a POISON CENTER/doctor.
If exposed: Call a poison center/doctor
Store in well-ventilated place. Keep cool. Store locked up. Keep container tightly closed.
Static accumulating flammable liquid can become electrostatically charged even in bonded and grounded equipment.
Sparks may ignite liquid and vapor. May cause flash fire or explosion. Use suitable extinguishing media to extinguish