BD, 560746, BD Phosflow™ Perm Buffer IV 10×

Preparation And Storage: Preparation And Storage Store undiluted at room temperature.
Recommended Assay Procedures: Recommended Assay Procedures This is the standard protocol for permeabilizing fixed leukocytes with Perm Buffer IV for subsequent staining of cell-surface markers and phosphorylated signaling proteins. 1. Prewarm BD Cytofix™ Fixation Buffer (Cat. No. 554655), for PBMC, or BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049), for whole blood or splenocytes, to 37°C prior to use. 2. Dilute Perm Buffer IV to 1.0× using 1× Phosphate Buffered Saline (PBS) prior to use. 3. Prepare cells. Use step a for PBMC or step b for whole blood or splenocytes. a. Isolate PBMC by density gradient separation (eg, using BD Vacutainer® CPT Cell Preparation Tube with Sodium Heparin, Cat. No. 362753) of whole blood, wash the cells well, and then treat them with the desired activator, inhibitor or combinations thereof. At the end of the cell treatment, immediately mix one volume of the warmed BD Cytofix Fixation Buffer with one volume of the suspended PBMC. Mix well and incubate the tubes in a 37°C water bath for 10 to 15 minutes. Spin down the cells at 400 g for 10 minutes in a table-top centrifuge. Aspirate the supernatant. or b. Obtain whole blood or suspend spleen cells. Treat the cells (eg, 100 µL) with the desired activator and/or inhibitor. After treatment, add a 20-fold excess volume (eg, 2.0 ml) of warmed BD Phosflow™ Lyse/Fix Buffer. Mix well and incubate the tubes in a 37°C water bath for 10 to 15 minutes. Spin down the cells at 400 g for 10 minutes in a table-top centrifuge. Aspirate the supernatant. 4. Wash the fixed leukocytes once with PBS. Pellet the cells by centrifugation at 400 g and remove all the supernatant. Note: High residual volumes of supernatant left over after the centrifugation and aspiration steps may dilute the Perm Buffer IV that is subsequently added. This may result in poor staining profiles. 5. Vortex the tubes to loosen the cells. Permeabilize the cells by slowly adding Perm Buffer IV dropwise to the cells. Add approximately 10 ml of Perm Buffer IV per 5 to 10 million cells.  Add a minimum of 1ml of Perm Buffer IV for cell concentration less than 5 million cells. Incubate at room temperature for 15 to 20 minutes. Note 1: Longer permeabilization time or using a ratio of cell to buffer volume outside the recommended ratio may result in poorer   fluorescent surface marker- and/or phosphorylated protein-specific antibody staining and detection. Note 2: Permeabilization with Perm Buffer IV may result in decreased cell recovery. 6. Centrifuge at 400 g for 10 minutes and remove the supernatant by aspiration. 7. Wash twice by adding BD Pharmingen™ Stain Buffer (FBS) to the cells. Centrifuge at 400 g for 10 minutes and remove the supernatant by aspiration each time. 8. Resuspend the cells in BD Pharmingen™ Stain Buffer (FBS) at 10X10^6 cells/ml and aliquot 100 µl to each sample tube to continue with antibody staining and flow cytometric analysis. It should also be noted that if staining of cell surface markers after cellular permeabilization with Perm Buffer IV does not work well, then the cells can be pre-stained with fluorescent antibodies directed against surface markers prior to cellular fixation or between the fixation and permeabilization steps. Staining can then be performed with antibodies specific for phosphorylated intracellular signaling molecules.
Product Notices: Product Notices Please refer to www.bdbiosciences.com/us/s/resources for technical protocols. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).