BD, 554654, BD Pharmingen™ MiCK-3 Mouse Cytokine Positive Control Cells

Application: Intracellular staining (flow cytometry) (Routinely Tested)
Concentration: 5x10^6 cells/ml
RRID: AB_2869004
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. Upon receipt, store the cell suspension at -80°C. Alternatively, the frozen cell suspension can be thawed and the capped vial should be quick spun to ensure complete retrieval of the contents from the vial. After thoroughly resuspending cells with a pipette, “single-use” aliquots can be refrozen and stored (-80°C) in polypropylene microtubes for a later time.
Recommended Assay Procedures: Recommended Assay Procedures MiCK-3 Positive Control Cell suspensions are intended to provide cells that contain intracellular accumulations of IL-1α, IL-6, IL-12, MCP-1 or TNF which are easily detectable by immunofluorescent staining of intracellular cytokines and flow cytometry. As such, these cells serve as positive controls for verifying the binding activity of fluorescent anti-cytokine antibodies and the staining procedure itself (e.g., permeabilization). For 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. The fixed and non-permeabilized cells should be washed twice with staining buffer to remove the DMSO.  The cells must then be incubated for 10 min followed by one wash in permeabilization/wash buffer (BD Perm/Wash™ Cat. No. 554723). The cells may then be stained with a fluorescent conjugate of either ALF-161 (hamster anti-mouse IL-1α; Cat. No. 559810), MP5-20F3 (rat anti-mouse IL-6; Cat. No. 554401), C15.6 (rat anti-mouse IL-12 p40/p70; Cat. No. 554479, or No. 554480), 2H5 (hamster anti-mouse MCP-1; Cat. No. 554443), MP6-XT22 (rat anti-mouse TNF; Cat. No. 554418, No. 554419 or No. 554420). Note: Cytokine-specific staining on MiCK-3 cells can be demonstrated by preincubation of conjugated cytokine-specific antibody with recombinant cytokine or by pretreatment of the MiCK-3 cells with unlabled blocking antibody.  Due to the cell activation procedure, a small proportion of cells may stain non-specifically (i.e., not blocked with either unlabeled antibody or ligand).