The 2F1 monoclonal antibody specifically binds to KLRG1 (Killer cell Lectin-like Receptor G1), which is the mouse homolog of the rat mast cell function-associated antigen (MAFA), on all mouse strains tested (eg, AKR/J, BALB/c, C3H/HeN, C3H.SW, C57BL/6, DBA/1, SJL, 129/J). Unlike rat MAFA, which is expressed on mast cells, mouse KLRG1 is expressed on a large subset of NK cells, lymphokine-activated killer (LAK) cells, adherent LAK (A-LAK) cells, subsets of activated CD8+ T lymphocytes, and small fractions of CD4+ and CD8+ T cells, but not mast cells. The expression of KLRG1 is correlated with reduced proliferative capacity of activated T lymphocytes or reduced effector functions of activated NK cells. KLRG1 plays a role in the regulation of leucocytes of both the innate and adaptive immune system. The 2F1 mAb reportedly stains the rat basophilic leukemia cell line, RBL-2H3, which is known to express MAFA. The KLRG1 protein is an inhibitory lectin-like type II transmembrane receptor containing a cytoplasmic motif similar to ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif); its ligand has not been identified KLRG1 is expressed mainly as a homodimeric molecule consisting of two N-glycosylated subunits of approximately 30-38 kDa. The level of KLRG1 expression is reduced in MHC class I-deficient mice, although direct binding of KLRG1 to MHC class I antigens could not be detected. Crosslinking of KLRG1 by 2F1 mAb reduces TCR-mediated Ca++ mobilization and cytotoxic responses (but not IFN-γ production) by CD8+ T cells and inhibits IFN-γ and TNF production and redirected lysis by 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).
3. For U.S. patents that may apply, see bd.com/patents.
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. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
11. 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.
12. Tandem fluorochromes contain both an energy donor and an energy acceptor. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from the donor may be observed. Additionally, multi-laser cytometers may directly excite both the donor and acceptor fluorochromes. Therefore, we recommend for every tandem conjugate, a matched individual single-stain control be acquired for generating a compensation or spectral unmixing matrix.
