Two-color flow cytometric analysis of Ly-6C expression on Mouse splenic leukocytes. BALB/c Mouse splenocytes were treated with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899) to lyse erythrocytes, washed, and preincubated with Purified Rat Anti-Mouse CD16/32 antibody (Mouse Fc Block™) [Cat. No. 553141/553142]. The cells were then stained with BD Horizon™ BUV395 Rat Anti-Mouse CD8a antibody (Cat. No. 563786) and with either BD Horizon™ RB613 Rat IgM, κ Isotype Control (Cat. No. 571105; Left Plot) or BD Horizon™ RB613 Rat Anti-Mouse Ly-6C antibody (Cat. No. 571103/571104; Right Plot) at 1 μg/test. The bivariate pseudocolor density plot showing the correlated expression of Ly-6C (or Ig Isotype control staining) versus CD8a was derived from gated events with the forward and side light-scatter characteristics of viable splenic leukocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ Software. Data shown on this Technical Data Sheet are not lot specific.
Two-color flow cytometric analysis of Ly-6C expression on Mouse splenic leukocytes. BALB/c Mouse splenocytes were treated with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899) to lyse erythrocytes, washed, and preincubated with Purified Rat Anti-Mouse CD16/32 antibody (Mouse Fc Block™) [Cat. No. 553141/553142]. The cells were then stained with BD Horizon™ BUV395 Rat Anti-Mouse CD8a antibody (Cat. No. 563786) and with either BD Horizon™ RB613 Rat IgM, κ Isotype Control (Cat. No. 571105; Left Plot) or BD Horizon™ RB613 Rat Anti-Mouse Ly-6C antibody (Cat. No. 571103/571104; Right Plot) at 1 μg/test. The bivariate pseudocolor density plot showing the correlated expression of Ly-6C (or Ig Isotype control staining) versus CD8a was derived from gated events with the forward and side light-scatter characteristics of viable splenic leukocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ Software. Data shown on this Technical Data Sheet are not lot specific.
Product Details
BD Horizon™
Ly6c; Lymphocyte antigen 6 complex, locus C; Lymphocyte antigen Ly-6C
Mouse (QC Testing)
Rat IgM, κ
Not reported
Flow cytometry (Routinely Tested)
0.2 mg/ml
17067
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions and unreacted dye was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- 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.
- Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
- 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.
- CF™ is a trademark of Biotium, Inc.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- For U.S. patents that may apply, see bd.com/patents.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) RUO
Size
0.5 mg
Cat No.
553142
RB613 Rat IgM, κ Isotype Control RUO
Size
0.1 mg
Cat No.
571105
BUV395 Rat Anti-Mouse CD8a RUO
Size
50 µg
Cat No.
563786
Lysing Buffer RUO
Size
100 mL
Cat No.
555899
571104 Rev. 1
Antibody Details
AL-21
The AL-21 monoclonal antibody specifically binds to a non-polymorphic determinant of Ly-6C, a 14-17 kDa GPI-linked cell-surface antigen found on some monocyte/macrophage populations, granulocytes, endothelial cells, plasma cells, and thymocyte, NK-cell, and T-subsets. Mice with the Ly-6.2 alloantigen (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.
571104 Rev. 1
Format Details
RB613
The BD Horizon RealBlue™ 613 (RB613) Dye is part of the BD® family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 492-nm and an emission maximum (Em Max) at 613-nm as measured using an antibody-dye conjugate. Driven by BD® innovation, RB613 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with reduced excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB613 can be used as an alternative to PE-CF594 and we recommend using an optical filter centered near 610-nm (eg, a 610/20-nm bandpass filter). For spectral instruments equipped with a Blue laser (488-nm), it can be used in conjunction with PE-CF594. RB613 is on average brighter than PE-CF594 off the blue laser.
RB613
492 nm
613 nm
571104 Rev.1
Citations & References
View product citations for antibody "571104" on CiteAb
Development References (7)
-
Cerwenka A, Carter LL, Reome JB, Swain SL, Dutton RW. In vivo persistence of CD8 polarized T cell subsets producing type 1 or type 2 cytokines. J Immunol. 1998; 161(1):97-105. (Biology). View Reference
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Jutila DB, Kurk S, Jutila MA.. Differences in the expression of Ly-6C on neutrophils and monocytes following PI-PLC hydrolysis and cellular activation.. Immunol Lett. 1994 ; 41(1):49-57. (Biology). View Reference
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Jutila MA, Kroese FG, Jutila KL, et al. Ly-6C is a monocyte/macrophage and endothelial cell differentiation antigen regulated by interferon-gamma. Eur J Immunol. 1988; 18(11):1819-1826. (Biology). View Reference
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Sato N, Yahata T, Santa K. Functional characterization of NK1.1 + Ly-6C+ cells. Immunol Lett. 1996; 1(54):1-5-9. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
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Takahama Y, Sharrow SO, Singer A. Expression of an unusual T cell receptor (TCR)-V beta repertoire by Ly-6C+ subpopulations of CD4+ and/or CD8+ thymocytes. Evidence for a developmental relationship between Ly-6C+ thymocytes and CD4-CD8-TCR-alpha beta+ thymocytes. J Immunol. 1991; 147(9):2883-2891. (Clone-specific: Flow cytometry). View Reference
-
Tough DF, Borrow P, Sprent J. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science. 1996; 272(5270):1947-1950. (Biology). View Reference
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Wrammert J, Källberg E, Agace WW, Leanderson T. Ly6C expression differentiates plasma cells from other B cell subsets in mice. Eur J Immunol. 2002; 32(1):97-103. (Clone-specific: Flow cytometry). View Reference
571104 Rev. 1
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.