Description
The 16a11 monoclonal antibody specifically recognizes the Mouse CD159a alloantigen that is also known as NKG2A alloantigen (NKG2A[B6] or NKG2AB6) which is expressed on subsets of C57BL/6 mouse NK, NK-T, or activated CD8+ T cells. CD159a (NKG2AB6) is an ~40 kDa single-pass type II transmembrane glycoprotein that is encoded by Klrc1 (Killer cell lectin-like receptor subfamily C member 1). This NKG2 receptor is comprised of an extracellular region with one C-type lectin domain followed by a transmembrane segment and a cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). It is expressed on the cell surface as a heterodimer that is disulfide bonded to CD94, an invariant type II C-type lectin-like transmembrane glycoprotein. The 16a11 antibody does not recognize NKG2A[BALB], the NKG2A alloantigen expressed by BALB/c mouse leucocytes. This antibody neither crossreacts with other NKG2 family members, NKG2C (NKG2C[B6]) nor NKG2E (NKG2E[B6]) alloantigens, nor CD94. Heterodimeric complexes of CD94 with either NKG2A, C, or E recognize Qa-1, a non-classical self-MHC class I antigen, presenting the Qdm signal peptide. This recognition endows NK cells with the capacity to detect cells with abnormal MHC class I expression that may result from cellular transformation or viral infection. Ligand-bound CD159a (NKG2AB6):CD94 can play a role in the inhibition of NK cell-mediated target cell lysis. When costaining cells with the 20d5 monoclonal antibody that is specific for mouse NKG2A/C/E receptors, crossblocking was reportedly minimized by first incubating cells with the 16a11 antibody and followed by the 20d5 antibody.
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.
