BD, 555062, BD Pharmingen™ HiCK-2 Human Cytokine Positive Control Cells

Application: Intracellular staining (flow cytometry) (Routinely Tested)
Concentration: 5x10^6 cells/ml
RRID: AB_2869016
Storage Buffer: Frozen in FBS and 10% DMSO.
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage Store product at -80°C prior to use or for long term storage of stock solutions. Rapidly thaw and quick-spin product prior to use. Avoid multiple freeze-thaws of product. This preparation contains no preservatives, thus it should be handled under aseptic conditions. After thawing and thoroughly resuspending cells with a pipette, “single-use” aliquots can be refrozen at -80°C and stored in polypropylene microtubes for use at a later time.
Recommended Assay Procedures: Recommended Assay Procedures Flow Cytometry: HiCK-2 Cytokine Positive Control Cell suspensions contain intracellular IL-3, IL-4, IL-10, IL-13 and GM-CSF which have accumulated and are detectable by intracellular flow cytometric analysis. These cells may serve as a positive control for verifying anti-cytokine antibody performance and/or the flow cytometric staining procedure itself (e.g. permeabilization).  For flow cytometric staining, the frozen cell preparation should first be quickly and carefully thawed.  Aliquots of the cell suspension can then be transferred to microwells or tubes.  HiCK-2 cells are supplied fixed and non-permeabilized in dimethylsulfoxide (DMSO), so should be washed at least twice with staining buffer to remove the DMSO. The cells must then be permeabilized by incubating for 10-15 min in BD Perm/Wash™ buffer (Cat. No. 554723), spun down to pellet and then followed by at least one wash in BD Perm/Wash™ buffer. Cells can then be stained with either PE Rat Anti-Human IL-3 (Cat. No. 554676), PE Mouse Anti-Human IL-4 (Cat. No. 554516), PE Rat Anti-Human IL-10 (Cat. No. 554706), PE Rat Anti-Human IL-13 (Cat. No. 554571) or PE Rat Anti-Human GM-CSF (Cat. No. 554507).  Investigators should note that for optimal detection of IL-10 or IL-13, ≥ 20,000 events should be acquired on the flow cytometer. Note: Cytokine-specific antibody staining of HiCK-2 cells can be demonstrated by preincubation of conjugated cytokine-specific antibody with recombinant cytokine or by pretreatment of the HiCK-2 cells with unlabeled (purified) blocking antibody.  Investigators should note that variation with cell activation may contribute to suboptimal blocking.