1. Warm BD Phosflow™ Fix Buffer I (Cat. No. 557870) to 37 °C prior to use.
2. Chill BD Phosflow™ Perm Buffer III 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 III 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 III contains 87.68% methanol (w/w).
Highly flammable liquid and vapor.
Toxic if swallowed, in contact with skin or if inhaled.
Causes damage to the central nervous system. Route of exposure: Oral.
Wear protective clothing / eye protection.
Wear protective gloves.
Take off immediately all contaminated clothing. Wash contaminated clothing before reuse.
Wash thoroughly after handling.
Use only outdoors or in a well-ventilated area.
Keep away from heat/sparks/open flames/hot surfaces. - No smoking.
Ground and bond container and receiving equipment. Use explosion-proof [electrical/ventilating/lighting/] equipment.
Use non-sparking tools. Take action to prevent static discharges.
Do not breathe mist/vapours/spray.
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.
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.
If exposed: Call a poison center/doctor.
In case of fire: Use suitable extinguishing media to extinguish.
Store in well-ventilated place. Keep cool. Store locked up. Keep container tightly closed.