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.
For optimal and reproducible results, BD Horizon Brilliant™ Stain Buffer should be used anytime BD Horizon Brilliant™ dyes are used in a multicolor flow cytometry panel. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. When BD Horizon Brilliant Stain Buffer is used in in the multicolor panel, it should also be used in the corresponding compensation controls for all dyes to achieve the most accurate compensation. For the most accurate compensation, compensation controls created with either cells or beads should be exposed to BD Horizon Brilliant Stain Buffer for the same length of time as the corresponding multicolor panel. 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
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- BD Horizon Brilliant Blue 700 is covered by one or more of the following US patents: 8,455,613 and 8,575,303.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
- An isotype control should be used at the same concentration as the antibody of interest.
Companion Products
The rat anti-mouse CD49b monoclonal antibody (clone DX5) specifically binds to the integrin α2 chain (CD49b). CD49b is a 150 kDa transmembrane glycoprotein that non-covalently associates with CD29 (integrin β1) to form the integrin α2β1 complex known as VLA-2. The rat anti-mouse CD49b antibody (clone DX5) has been reported to identify the majority of NK cells and a small T-cell subpopulation in most mouse strains (e.g., A/J, AKR, BALB/c, C3H/HeJ, C57BL/6, C57BL/10, C57BR, C58, CBA/Ca, DBA/1, DBA/2, SJL, SWR, 129/J, but not NOD). The DX5 antibody also recognizes platelets that express high levels of CD49b. Multiparameter flow cytometric analysis has demonstrated that most lymphocytes which express NK-1.1 (NKR-P1B and NKR-P1C), as detectable by mouse anti-mouse NK-1.1 antibody (clone PK136), also express the DX5 antigen. Small DX5+ NK-1.1- and DX5- NK-1.1+ cell subsets are found, especially among the CD3-positive cell population. Some CD49b+ NK cells have been reported to gradually lose reactivity with the rat anti-mouse CD49b antibody (clone DX5) when cultured in the presence of recombinant human IL-2. The resulting DX5-negative cells have weakened cytotoxic activity when compared to the remaining DX5+ cells. This indicates that the DX5 antibody distinguishes functional subsets of NK cells. No activation or blocking activity of the rat anti-mouse antibody (clone DX5) has been observed. Staining of splenic NK cells with this antibody reportedly can be blocked by hamster anti-mouse CD49b antibody (clone HMα2).
Development References (3)
-
Arase H, Saito T, Phillips JH, Lanier LL. Cutting edge: the mouse NK cell-associated antigen recognized by DX5 monoclonal antibody is CD49b (alpha 2 integrin, very late antigen-2). J Immunol. 2001; 167(3):1141-1144. (Clone-specific: Blocking, Cytotoxicity, Flow cytometry). View Reference
-
Moore TA, von Freeden-Jeffry U, Murray R, Zlotnik A. Inhibition of gamma delta T cell development and early thymocyte maturation in IL-7 -/- mice. J Immunol. 1996; 157(6):2366-2373. (Biology). View Reference
-
Ortaldo JR, Winkler-Pickett R, Mason AT, Mason LH. The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells. J Immunol. 1998; 160(1):1158-1165. (Biology). View Reference
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
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.