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Multiparameter flow cytometric analysis of CD172a/b expression on human peripheral blood leucocyte populations. Human whole blood was stained with either BD Horizon™ R718 Mouse IgG1, κ Isotype Control (Cat. No. 566928; Left Plot) or BD Horizon™ R718 Mouse Anti-Human CD172a/b antibody (Cat. No. 567088; Right Plot). Erythrocytes were lysed with BD FACS ™ Lysing Solution (Cat. No. 349202). Bivariate pseudocolor density plots showing the correlated expression of CD172a/b [or Ig Isotype control staining] versus side light-scatter (SSC-A) signals were derived from gated events with the forward and side light-scatter characteristics of intact leucocyte populations. Flow cytometry and data analysis was performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
BD Horizon™ R718 Mouse Anti-Human CD172a/b
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Preparation And Storage
Recommended Assay Procedures
BD® CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to BD® 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 cells and BD CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
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The SE5A5 monoclonal antibody specifically binds to a common epitope on CD172a/SIRPα (90 kDa) and CD172b/SIRPβ1 (50 kDa). These transmembrane glycoproteins are members of the Signal Regulatory Protein (SIRP) family that, in turn, belongs to the Immunoglobulin superfamily. The SIRP family is comprised of two subgroups, SIRPα and SIRPβ that are distinguished by the presence (α) or absence (β) of a cytoplasmic domain containing two immunoreceptor tyrosine-based inhibition motifs (ITIM). CD172a/SIRPα is expressed on CD34+ stem/progenitor cells, cardiomyocytes, monocytes, macrophages, granulocytes, dendritic cells, and in the central nervous system. It binds to CD47 and is implicated in mediating inhibitory signals via the ITIM/SHP-2 association. CD172b/SIRPβ1 does not possess a cytoplasmic domain but instead the transmembrane domain contains a positively-charged residue that can interact with another transmembrane protein, DAP-12/KARAP. DAP-12 has two immunoreceptor tyrosine-based activation motifs (ITAM) within its cytoplasmic domain that are thought to link CD172b to cellular activation signaling. CD172b is expressed on myeloid cells, including peripheral blood monocytes and granulocytes. It is not expressed on CD34+ cells. CD172a and CD172b have complementary roles in signal regulation and may work together in tuning certain cellular responses to stimuli.
The antibody was conjugated to BD Horizon™ Red 718, which has been developed exclusively by for BD Biosciences as a better alternative to Alexa Fluor™ 700. BD Horizon™ Red 718 can be excited by the red laser (628 – 640 nm) and, with an Em Max around 718 nm, it can be detected using a 730/45 nm filter. Due to similar excitation and emission properties, we do not recommend using R718 in combination with APC-R700 or Alexa Fluor™ 700.
Development References (7)
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Bühring HJ, Simmons DL, Vernon-Wilson E. Review—CD172—SIRP; signal regulatory protein. In: Mason D. David Mason .. et al., ed. Leucocyte typing VII : white cell differentiation antigens : proceedings of the Seventh International Workshop and Conference held in Harrogate, United Kingdom. Oxford: Oxford University Press; 2002:35.
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Dietrich J, Cella M, Seiffert M, Bühring HJ, Colonna M. Cutting edge: signal-regulatory protein beta 1 is a DAP12-associated activating receptor expressed in myeloid cells. J Immunol. 2000; 164(1):9-12. (Biology). View Reference
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Dubois NC, Craft AM, Sharma P, et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat Biotechnol. 2011; 29:1011-1018. (Biology). View Reference
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Ghannadan M, Hauswirth AW, Schernthaner GH, et al. Detection of novel CD antigens on the surface of human mast cells and basophils. Int Arch Allergy Immunol. 2002; 127(4):299-307. (Biology). View Reference
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Seiffert M, Brossart P, Cant C, et al. Signal-regulatory protein alpha (SIRPalpha) but not SIRPbeta is involved in T-cell activation, binds to CD47 with high affinity, and is expressed on immature CD34(+)CD38(-) hematopoietic cells.. Blood. 2001; 97(9):2741-9. (Clone-specific: Immunoprecipitation, Inhibition). View Reference
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Seiffert M, Cant C, Chen Z, et al. Human signal-regulatory protein is expressed on normal, but not on subsets of leukemic myeloid cells and mediates cellular adhesion involving its counterreceptor CD47. Blood. 1999; 94(11):3633-3643. (Immunogen: Flow cytometry, Functional assay, Immunoprecipitation, Inhibition). View Reference
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Simmons DL, Vernon-Wilson E. Structure and function of the signal regulatory proteins (SIRPs). In: Mason D. David Mason .. et al., ed. Leucocyte typing VII : white cell differentiation antigens : proceedings of the Seventh International Workshop and Conference held in Harrogate, United Kingdom. Oxford: Oxford University Press; 2002:35-38.
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.