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 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).
For optimal results, it is recommended to perform two washes after staining with antibodies. Cells may be prepared, stained with antibodies and washed twice with wash buffer per established protocols for immunofluorescent staining prior to acquisition on a flow cytometer. Performing fewer than the recommended wash steps may lead to increased spread of the negative population.
Product Notices
- The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.
- Researchers should determine the optimal concentration of this reagent for their individual applications.
- 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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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 http://regdocs.bd.com to access safety data sheets (SDS).
- BD Horizon Brilliant Blue 700 is covered by one or more of the following US patents: 8,455,613 and 8,575,303.
- Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
Companion Products
The CG4.rMab is a recombinant monoclonal antibody that was derived from CG4 hybridoma cells. The CG4.rMab specifically binds to human G-protein coupled receptor 56 (GPR56) like the conventional CG4 antibody and performs like the CG4 antibody when used to stain cells and analyze them by flow cytometry. GPR56 is also known as adhesion G-protein coupled receptor G1 (ADGRG1), or TM7XN1. GPR56 is a ~15kDa G protein-coupled receptor encoded by ADGRG1 which belongs to the adhesion-GPCR family that comprises 33 members in human. The extracellular region contains a mucin-like domain followed by a membrane proximal GPCR-autoproteolysis inducing (GAIN) domain, seven transmembrane regions and a cytoplasmic tail. The constitutive self-cleavage at the proteolytic site gives rise to a membrane spanning (C-terminal fragment or CTF) and an extracellular (N-terminal fragment or NTF) subunit that remain noncovalently bound, leading to the expression of a heterodimeric receptor at the cell surface. GPR56 is widely expressed with the highest levels of messenger found in the brain, heart, and thyroid gland. Recently, GPR56 was found to be variably expressed on platelets, cytotoxic NK cells and T lymphocytes including CD4+, CD8+, and γδ T cells. It was shown that GPR56 functions as an inhibitory receptor on NK cells through interaction with CD81. While GPR56 NTF associates with Tissue transglutaminase 2 and Collagen III (α-1), GPR56 CTF can recruit Gα proteins leading to the activation of mTOR and RhoA signaling pathways. GPR56 has been implicated in cell-cell interactions, adhesion, migration, and regulation of cell proliferation and survival of various cell types. New evidence also shows a role of GPR56 in tumor progression. Recently, the CG4 antibody was found to activate GPR56 in melanoma cells leading to an increase of IL-6 secretion, in a CD9/CD81-dependent manner.
The antibody was conjugated to BD Horizon BB700, which is part of the BD Horizon Brilliant™ Blue family of dyes. It is a polymer-based tandem dye developed exclusively by BD Biosciences. With an excitation max of 485 nm and an emission max of 693 nm, BD Horizon BB700 can be excited by the 488 nm laser and detected in a standard PerCP-Cy™5.5 set (eg, 695/40-nm filter). This dye provides a much brighter alternative to PerCP-Cy5.5 with less cross laser excitation off the 405 nm and 355 nm lasers.
Development References (6)
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Della Chiesa M, Falco M, Parolini S, et al. GPR56 as a novel marker identifying the CD56dull CD16+ NK cell subset both in blood stream and in inflamed peripheral tissues.. Int Immunol. 2010; 22(2):91-100. (Biology: Flow cytometry). View Reference
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Liu M, Parker RM, Darby K, et al. GPR56, a novel secretin-like human G-protein-coupled receptor gene.. Genomics. 1999; 55(3):296-305. (Biology). View Reference
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Pabst C, Bergeron A, Lavallée VP, et al. GPR56 identifies primary human acute myeloid leukemia cells with high repopulating potential in vivo.. Blood. 2016; 127(16):2018-27. (Clone-specific: Flow cytometry). View Reference
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Peng YM, van de Garde MD, Cheng KF, et al. Specific expression of GPR56 by human cytotoxic lymphocytes.. J Leukoc Biol. 2011; 90(4):735-40. (Immunogen: Flow cytometry). View Reference
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Piao X, Hill RS, Bodell A, et al. G protein-coupled receptor-dependent development of human frontal cortex.. Science. 2004; 303(5666):2033-6. (Biology). View Reference
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Rao TN, Marks-Bluth J, Sullivan J, et al. High-level Gpr56 expression is dispensable for the maintenance and function of hematopoietic stem and progenitor cells in mice.. Stem Cell Res. 2015; 14(3):307-22. (Clone-specific: Flow cytometry). View Reference
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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.