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 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 CompBead to ensure that BD Comp beads are appropriate for your specific cellular application.
For optimal results, it is recommended to perform 2 washes after staining with antibodies. Cells may be prepared, stained with antibodies and washed twice with wash buffer per established protocols for immunofluorescence staining, prior to acquisition on a flow cytometer. Performing fewer than the recommended wash steps may lead to increased spread of the negative population.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
The M1/70 monoclonal antibody specifically binds to CD11b, also known as Integrin alpha M (Itgam or αM). CD11b is a 170-kDa type 1 transmembrane glycoprotein and belongs to the Integrin alpha chain family. CD11b serves as the alpha chain of the heterodimeric Mac-1 integrin (CD11b/CD18, αMβ2), also known as complement receptor 3 (CR3). Mac-1 mediates adhesion to ICAM-1 (CD54), ICAM-2 (CD102), fibrinogen and binding to C3bi. Mac-1 is expressed at varying levels on granulocytes, macrophages, myeloid-derived dendritic cells, natural killer cells, microglia, and B-1 B lymphocytes. Mac-1 expression is rapidly upregulated on neutrophils after activation, in the same time period that CD62L (L-selectin) is shed from the cell surface. The M1/70 antibody reportedly blocks cell adherence and C3bi binding but does not block cell-mediated lysis. Cross-reaction of the M1/70 antibody with CD11b expressed on human monocytes, polymorphonuclear leukocytes, and NK cells has been reported.
The antibody was conjugated to BD Horizon BB515 which is part of the BD Horizon Brilliant™ Blue family of dyes. With an Ex Max near 490 nm and an Em Max near 515 nm, BD Horizon BB515 can be excited by the blue laser (488 nm) laser and detected with a 530/30 nm filter. This dye has been exclusively developed by BD Biosciences and is up to seven times brighter than FITC with less spillover into the PE channel. Due to similar excitation and emission properties, BB515, FITC, and Alexa Fluor® 488 cannot be used simultaneously. It is not recommended to use BB515 in cocktails that include Streptavidin conjugates as it may cause high background.
Development References (12)
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Ault KA, Springer TA. Cross-reaction of a rat-anti-mouse phagocyte-specific monoclonal antibody (anti-Mac-1) with human monocytes and natural killer cells. J Immunol. 1981; 126(1):359-364. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting, Radioimmunoassay). View Reference
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Beller DI, Springer TA, Schreiber RD. Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor. J Exp Med. 1982; 156(4):1000-1009. (Clone-specific: Blocking, Inhibition). View Reference
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Driver DJ, McHeyzer-Williams LJ, Cool M, Stetson DB, McHeyzer-Williams MG. Development and maintenance of a B220- memory B cell compartment. J Immunol. 2001; 167(3):1393-1405. (Clone-specific: Flow cytometry, Immunofluorescence). View Reference
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Kaji K, Takeshita S, Miyake K, Takai T, Kudo A. Functional association of CD9 with the Fc gamma receptors in macrophages. J Immunol. 2001; 166(5):3256-3265. (Clone-specific: Fluorescence microscopy, Immunofluorescence). View Reference
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Kishimoto TK, Jutila MA, Berg EL, Butcher EC. Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors. Science. 1989; 245(4923):1238-1241. (Biology). View Reference
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Lagasse E, Weissman IL. Flow cytometric identification of murine neutrophils and monocytes. J Immunol Methods. 1996; 197(1-2):139-150. (Clone-specific: Flow cytometry). View Reference
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Lub M, van Kooyk Y, Figdor CG. Competition between lymphocyte function-associated antigen 1 (CD11a/CD18) and Mac-1 (CD11b/CD18) for binding to intercellular adhesion molecule-1 (CD54). J Leukoc Biol. 1996; 59(5):648-655. (Clone-specific: Immunoprecipitation). View Reference
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Sanchez-Madrid F, Simon P, Thompson S, Springer TA. Mapping of antigenic and functional epitopes on the alpha- and beta-subunits of two related mouse glycoproteins involved in cell interactions, LFA-1 and Mac-1. J Exp Med. 1983; 158(2):586-602. (Clone-specific: Immunoaffinity chromatography, Immunoprecipitation, Inhibition). View Reference
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Springer T, Galfre G, Secher D, Milstein C. Monoclonal xenogeneic antibodies to mouse leukocyte antigens: identification of macrophage-specific and other differentiation antigens. Curr Top Microbiol Immunol. 1978; 81:45-50. (Immunogen: Immunoprecipitation, Radioimmunoassay). View Reference
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Springer T, Galfre G, Secher DS, Milstein C. Mac-1: a macrophage differentiation antigen identified by monoclonal antibody. Eur J Immunol. 1979; 9(4):301-306. (Clone-specific: Flow cytometry, Immunoprecipitation). View Reference
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Springer T, Galfre G, Secher DS, Milstein C. Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Eur J Immunol. 1978; 8(8):539-551. (Immunogen: Immunoprecipitation). View Reference
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Springer TA, Davignon D, Ho MK, Kurzinger K, Martz E, Sanchez-Madrid F. LFA-1 and Lyt-2,3, molecules associated with T lymphocyte-mediated killing; and Mac-1, an LFA-1 homologue associated with complement receptor function. Immunol Rev. 1982; 68:171-195. (Immunogen: Immunoprecipitation, Radioimmunoassay). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.