1. Collect normal human blood in the presence of EDTA or heparin and treat with appropriate activators where applicable.
2. Dilute the required amount of BD Phosflow™ Lyse/Fix Buffer (5X concentrate) 1:5 with deionoized or distilled water (at room temperature) and then pre-warm the solution to 37°C. The 1X working solution should be made fresh for each experiment and any remaining solution at the end of the experiment should be discarded.
3. Mix one volume of blood with 20 volumes of the pre-warmed BD Phosflow™ Lyse/Fix Buffer (1X). Mix well by vigorously inverting the tubes 8-10 times, and then incubate the tubes in a 37°C water bath for 10 min.
4. Spin down the cells at 500x g for 8 min in a table-top centrifuge, aspirate the supernatant, and wash the cells once with Hank's solution.
5. Vortex to loosen the cells and permeablize the cells by adding the appropriate permeabilization reagent, such as cold BD Phosflow™ Perm Buffer II, slowly while vortexing. Incubate on ice for 30 min and then spin down the cells.
6. Wash the cells twice and resuspend with BD Pharmingen™ Staining Buffer.
7. Stain the cells with appropriate antibodies for 30-60 min at room temperature, wash and prepare for flow cytometric analysis.
Danger: BD Phosflow™ Lyse/Fix Buffer contains 7.15% methanol (w/w), 20.35% formaldehyde (w/w) and 15.65% diethylene glycol (w/w).
Harmful if swallowed or in contact with skin.
Toxic if inhaled.
Causes skin irritation.
Causes serious eye damage.
May cause an allergic skin reaction.
Suspected of causing genetic defects.
May cause cancer. Route of exposure: Inhalative.
May cause damage to organs. May cause respiratory irritation.
May cause damage to the kidneys through prolonged or repeated exposure. Route of exposure: Oral.
Obtain special instructions before use.
Wear protective gloves / eye protection.Wear protective clothing.
Do not breathe mist/vapours/spray.
IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin with water/shower.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do.
IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing. Get immediate medical advice/attention.
IF exposed or concerned: Get medical advice/attention.
Store in a well-ventilated place. Keep container tightly closed.