BD Pharmingen™ Alexa Fluor™ 647 Rat Anti-Mouse Ly-6C
Clone HK1.4.rMAb (also known as HK1.4) (RUO)
Multicolor flow cytometric analysis of Ly-6C expression on Mouse splenic leucocytes.
Top Plots: BALB/c Mouse splenocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) [Cat. No. 553141/553142]. The cells were then stained with FITC Rat Anti-Mouse CD8a antibody (Cat. No. 553030/561966) and with either Alexa Fluor™ 647 Rat IgG1, κ Isotype Control (Cat. No. 557731; Left Plot) or Alexa Fluor™ 647 Rat Anti-Mouse Ly-6C antibody (Cat. No. 569434/569435; Right Plot) at 0.5 μg/test.
Bottom Plots: BALB/c Mouse splenocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™). The cells were then stained with BD Horizon™ BUV737 Rat Anti-CD11b antibody (Cat. No. 612800) and with either Alexa Fluor™ 647 Rat IgG1, κ Isotype Control (Left Plot) or Alexa Fluor™ 647 Rat Anti-Mouse Ly-6C antibody (Right Plot) at 0.5 μg/test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The bivariate pseudocolor density plots showing the correlated expression of Ly-6C (or Ig Isotype control staining) versus CD8a (Top Plots) or CD11b (Bottom Plots) were derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD-negative) leucocytes.
Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Multicolor flow cytometric analysis of Ly-6C expression on Mouse splenic leucocytes.
Top Plots: BALB/c Mouse splenocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) [Cat. No. 553141/553142]. The cells were then stained with FITC Rat Anti-Mouse CD8a antibody (Cat. No. 553030/561966) and with either Alexa Fluor™ 647 Rat IgG1, κ Isotype Control (Cat. No. 557731; Left Plot) or Alexa Fluor™ 647 Rat Anti-Mouse Ly-6C antibody (Cat. No. 569434/569435; Right Plot) at 0.5 μg/test.
Bottom Plots: BALB/c Mouse splenocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™). The cells were then stained with BD Horizon™ BUV737 Rat Anti-CD11b antibody (Cat. No. 612800) and with either Alexa Fluor™ 647 Rat IgG1, κ Isotype Control (Left Plot) or Alexa Fluor™ 647 Rat Anti-Mouse Ly-6C antibody (Right Plot) at 0.5 μg/test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The bivariate pseudocolor density plots showing the correlated expression of Ly-6C (or Ig Isotype control staining) versus CD8a (Top Plots) or CD11b (Bottom Plots) were derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD-negative) leucocytes.
Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
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 cell and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- 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.
- This product is provided under an intellectual property license between Life Technologies Corporation and BD Businesses. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for any other use, contact Life Technologies Corporation, Cell Analysis Business Unit Business Development, 29851 Willow Creek Road, Eugene, OR 97402, USA, Tel: (541) 465-8300. Fax: (541) 335-0504.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Alexa Fluor™ is a trademark of Life Technologies Corporation.
- For U.S. patents that may apply, see bd.com/patents.
Companion Products
The HK1.4.rMAb monoclonal antibody is a recombinant monoclonal antibody derived from HK1.4 hybridoma cells that specifically recognizes a non-polymorphic determinant on Mouse Lymphocyte antigen Ly-6C which is also known as Ly6c. Ly-6C is an ~14-17 kDa glycosylphosphatidylinositol (GPI)-linked cell-surface antigen that is encoded by Ly6c1 (Lymphocyte antigen 6 complex, locus C1) which belongs to the Ly-6 gene family. Ly-6C is expressed on monocytes, macrophages, neutrophils, eosinophils, endothelial cells, plasma cells, thymocytes, NK cells, and some T cell subsets. Mice with the Ly-6.2 haplotype (eg, AKR, C57BL, C57BR, C57L, C58, DBA/2, PL, SJL, SWR, 129) have subsets of CD8+ and CD4+ Ly-6C+ T cells, while Ly-6.1 strains (eg, A, BALB/c, CBA, C3H/He, DBA/1, NZB) have only CD8+ Ly-6C+ T cells. Upregulation of Ly-6C expression on CD8+ T cells by interferons α and β and poly (I:C) has been described, and Ly-6C is a memory marker on CD8+ T cells. The HK1.4 antibody does not reportedly block the binding of the RB6-8C5 monoclonal antibody that specifically recognizes Mouse Ly-6G and Ly-6C.
Development References (3)
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Havran WL, Lancki DW, Moldwin RL, Dialynas DP, Fitch FW. Characterization of an anti-Ly-6 monoclonal antibody which defines and activates cytolytic T lymphocytes.. J Immunol. 1988; 140(4):1034-42. (Immunogen: Activation, Blocking, (Co)-stimulation, Flow cytometry, Fluorescence activated cell sorting, Functional assay, Stimulation). View Reference
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Ma C, Kapanadze T, Gamrekelashvili J, Manns MP, Korangy F, Greten TF. Anti-Gr-1 antibody depletion fails to eliminate hepatic myeloid-derived suppressor cells in tumor-bearing mice.. J Leukoc Biol. 2012; 92(6):1199-206. (Clone-specific: Flow cytometry). View Reference
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Schlueter AJ, Malek TR, Hostetler CN, Smith PA, deVries P, Waldschmidt TJ. Distribution of Ly-6C on lymphocyte subsets: I. Influence of allotype on T lymphocyte expression.. J Immunol. 1997; 158(9):4211-22. (Biology). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.