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.
Bead-based compensation or unmixing controls, such as BD® CompBeads or BD™ SpectraComp™, can be used as surrogates to assess fluorescence spillover when bound to fluorochrome-conjugated antibodies. Although these beads have spectral properties similar to cells, variations in spectral emission may occur, resulting in differing spillover values compared to biological controls. Therefore, it is considered best practice to compare the spillover obtained from cells and bead-based controls when using BD® CompBeads or BD™ SpectraComp™ for the first time, to ensure they are appropriate for the intended application.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions that minimize unconjugated dye and antibody.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
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4. 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.
5. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
6. 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.
7. 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.
8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
9. An isotype control should be used at the same concentration as the antibody of interest.
10. 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.
11. Alexa Fluor™ is a trademark of Life Technologies Corporation.
