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Two-color analysis of NK-1.1 expression on lymphokine-activated killer (LAK) cells. LAK cells from C57BL/6 mice were stained with FITC-conjugated anti-mouse CD244.2 mAb 2B4 (Cat. No. 553305) and either PerCP-Cy5.5-conjugated mouse IgG2a κ isotype control mAb G155-178 (Cat. No. 551115, left panel) or PerCP-Cy5.5-conjugated mAb PK136 (right panel). Viable blasts were gated according to their light-scatter profile. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
BD Pharmingen™ PerCP-Cy™5.5 Mouse Anti-Mouse NK-1.1
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
PerCP has been reported to undergo significant photobleaching, the magnitudeof which increases as laser power is increased or beam focus is narrowed. For tandem conjugates incorporating PerCP (e.g., PerCP-Cy5.5), the excitation and emission properties of PerCP and the kinetics of energy exchange between the fluorochromes of the tandem dye may limit their effectiveness on high speed and/or sorting flow cytometers. Therefore, for third color flow cytometric analysis using ≥ 25-mW laser power, we recommend the PE-Cy7-conjugated reagent (Cat. No. 552878).
PerCP-Cy5.5 is a tandem fluorochrome composed of peridinin chlorophyll protein (PerCP), which is excited by the 488-nm line of an Argon ion laser and serves as the energy donor, coupled to the cyanine dye Cy5.5™, which acts as the energy acceptor and fluoresces at 695nm.
It is recommended that a 712/20-nm band-pass filter be used with stream-in-air instruments such as the BD FACStar™ and BD FACSVantage™ flow cytometry systems.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
- PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Cy is a trademark of Amersham Biosciences Limited. This conjugated product is sold under license to the following patents: US Patent Nos. 5,486,616; 5,569,587; 5,569,766; 5,627,027.
- This product is subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and made and sold under license from Amersham Biosciences Corp. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one return this material, unopened to BD Biosciences, 10975 Torreyana Rd, San Diego, CA 92121 and any money paid for the material will be refunded.
- 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.
Companion Products
In the mouse, at least three members of the Klrb (Killer cell lectin-like receptor, subfamily b; formerly NKR-P1) gene family have been identified (Klrb1a/NKR-P1A, Klrb1b/NKR-P1B, and Klrb1c/NKR-P1C); but in the human gene family, a single homologue has been designated KLRB1, NKR-P1A, or CD161. The KLRB1/NKR-P1 family of proteins are type-II-transmembrane C-type lectin receptors. KLRB1C/NKR-P1C activates NK-cell cytotoxicity, while KLRB1B/NKR-P1B functions as an inhibitory receptor. KLRB1B/NKR-P1B protein has intracellular Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM), while KLRB1C/NKR-P1C lacks ITIM and activates via association with Fc Receptor γ chain. Strikingly, KLRB1B/NKR-P1B and KLRB1C/NKR-P1C share 96% amino acid sequence identity in their extracellular C-type lectin domains. The PK136 antibody reacts with the NK-1.1 surface antigen (CD161c) encoded by the Klrb1c/NKR-P1C gene expressed on natural killer (NK) cells in selected strains of mice (eg, C57BL, FVB/N, NZB, but not A, AKR, BALB/c, CBA/J, C3H, C57BR, C58, DBA/1, DBA/2, NOD, SJL, 129) and the CD161b antigen encoded by the Klrb1b/NKR-P1B gene expressed only on Swiss NIH and SJL mice, but not on C57BL/6. Expression of KLRB1C/NKR-P1C protein is correlated with the ability to lyse tumor cells in vitro and to mediate rejection of bone marrow allografts. The NK-1.1 marker is useful in defining NK cells; however, the antigen is also expressed on a rare, specialized population of T lymphocytes (NK-T cells) and some cultured monocytes. Plate-bound PK136 mAb, in combination with low concentrations of IL-2, induces proliferation of a subset of NK cells.
Development References (5)
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Carlyle JR, Martin A, Mehra A, Attisano L, Tsui FW, Zuniga-Pflucker JC. Mouse NKR-P1B, a novel NK1.1 antigen with inhibitory function. J Immunol. 1999; 162(10):5917-5923. (Clone-specific: Immunoprecipitation). View Reference
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Greimers R, Trebak M, Moutschen M, Jacobs N, Boniver J. Improved four-color flow cytometry method using fluo-3 and triple immunofluorescence for analysis of intracellular calcium ion ([Ca2+]i) fluxes among mouse lymph node B- and T-lymphocyte subsets. Cytometry. 1996; 23(3):205-217. (Biology). View Reference
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Koo GC, Peppard JR. Establishment of monoclonal anti-Nk-1.1 antibody. Hybridoma. 1984; 3(3):301-303. (Immunogen: Cytotoxicity, Flow cytometry). View Reference
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Kung SK, Su RC, Shannon J, Miller RG. The NKR-P1B gene product is an inhibitory receptor on SJL/J NK cells. J Immunol. 1999; 162(10):5876-5887. (Clone-specific: Blocking). View Reference
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Reichlin A, Yokoyama WM. Natural killer cell proliferation induced by anti-NK1.1 and IL-2. Immunol Cell Biol. 1998; 76(2):143-152. (Clone-specific: Induction). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.