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Two-color flow cytometric analysis of KIR2DL1/S1/S3/S5 (CD158) expression on peripheral blood leucocyte populations. Whole blood was stained with BD Horizon™ PE-CF594 Mouse Anti-Human CD56 antibody (Cat. No. 564963) and either PE Mouse IgG2b, κ Isotype Control (Cat. No. 555058; Left Plots) or PE Mouse Anti-Human KIR2DL1/S1/S3/S5 (CD158) (Cat. No. 567158/567159; Right Plots). Erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). The bivariate pseudocolor density plots showing the corelated expression of KIR2DL1/S1/S3/S5 (CD158) [or Ig isotype control staining] versus side light scatter signals for leucocyte populations [Upper Plots] or CD56 for lymphocytes [Lower Plots] were derived from gated events with the forward and side-light scatter characteristics of viable leucocytes or lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Two-color flow cytometric analysis of KIR2DL1/S1/S3/S5 (CD158) expression on peripheral blood leucocyte populations. Whole blood was stained with BD Horizon™ PE-CF594 Mouse Anti-Human CD56 antibody (Cat. No. 564963) and either PE Mouse IgG2b, κ Isotype Control (Cat. No. 555058; Left Plots) or PE Mouse Anti-Human KIR2DL1/S1/S3/S5 (CD158) (Cat. No. 567158/567159; Right Plots). Erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). The bivariate pseudocolor density plots showing the corelated expression of KIR2DL1/S1/S3/S5 (CD158) [or Ig isotype control staining] versus side light scatter signals for leucocyte populations [Upper Plots] or CD56 for lymphocytes [Lower Plots] were derived from gated events with the forward and side-light scatter characteristics of viable leucocytes or lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Two-color flow cytometric analysis of KIR2DL1/S1/S3/S5 (CD158) expression on peripheral blood leucocyte populations. Whole blood was stained with BD Horizon™ PE-CF594 Mouse Anti-Human CD56 antibody (Cat. No. 564963) and either PE Mouse IgG2b, κ Isotype Control (Cat. No. 555058; Left Plots) or PE Mouse Anti-Human KIR2DL1/S1/S3/S5 (CD158) (Cat. No. 567158/567159; Right Plots). Erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). The bivariate pseudocolor density plots showing the corelated expression of KIR2DL1/S1/S3/S5 (CD158) [or Ig isotype control staining] versus side light scatter signals for leucocyte populations [Upper Plots] or CD56 for lymphocytes [Lower Plots] were derived from gated events with the forward and side-light scatter characteristics of viable leucocytes or lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
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BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cell and CompBead to ensure that BD Comp beads are appropriate for your specific cellular application.
The HP-MA4 monoclonal antibody specifically recognizes several Killer Cell Immunoglobulin-like Receptors (KIRs) which are also known as CD158 molecules. HP-MA4 recognizes Killer cell immunoglobulin-like receptor 2DL1 (encoded by KIR2DL1; aka, CD158a and NKAT-1), Killer cell immunoglobulin-like receptor 2DS1 (KIR2DS1; CD158h), Killer cell immunoglobulin-like receptor 2DS3 (KIR2DS3; NKAT-7), and Killer cell immunoglobulin-like receptor 2DS5 (KIR2DS5; CD158g, NKAT-9) which are collectively known as KIR2DL1/S1/S3/S5 (CD158). These type I transmembrane glycoproteins are encoded by polymorphic genes and have 2 extracellular Ig-like domains (KIR2D, domains D1 and D2) followed by a transmembrane region and either long (L) or short (S) cytoplasmic domains. Various CD158 molecules are differentially expressed by CD56dim natural killer (NK) cells and some T cells and can regulate their cytotoxic effector functions. Although different KIR gene content varies amongst haplotypes for individuals, certain "framework" genes including KIR3DL3, KIR3DP1, KIR3DL4, and KIR3DL2, are found in all haplotypes. KIR2DL1 has a long cytoplasmic domain with two tyrosine-based inhibitory motifs (ITIM) that enables inhibitory signal transduction by ligand-bound KIR2DL1 leading to reduced cytotoxic effector cell activity. KIR2DS1, KIR2DS3, KIR2DS5 (KIR2DS1/S3/S5) proteins each have a short cytoplasmic tail with a positively charged amino acid in their transmembrane region which allows association with the DAP12 transmembrane protein. DAP12 acts as an activating signal transduction element through its immunoreceptor tyrosine-based activation motifs (ITAMs) in its cytoplasmic domain leading to upregulated cytotoxic effector cell function. Some MHC class I molecules can serve as ligands for CD158 molecules, with HLA-C ligands reported for KIR2DL1, KIR2DS1, and KIR2DS5.
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