Preparation And Storage
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
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- For U.S. patents that may apply, see bd.com/patents.
- 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.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- 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.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- CF™ is a trademark of Biotium, Inc.
- 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.
Companion Products
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.
Development References (5)
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Joncker NT, Fernandez NC, Treiner E, Vivier E, Raulet DH. NK cell responsiveness is tuned commensurate with the number of inhibitory receptors for self-MHC class I: the rheostat model. J Immunol. 2009; 182(8):4572-4580. (Clone-specific: In vivo exacerbation). View Reference
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McMahon CW, Zajac AJ, Jamieson AM. Viral and bacterial infections induce expression of multiple NK cell receptors in responding CD8(+) T cells. J Immunol. 2002; 169(3):1444-1452. (Clone-specific: Flow cytometry). View Reference
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Vance RE, Jamieson AM, Cado D, Raulet DH. Implications of CD94 deficiency and monoallelic NKG2A expression for natural killer cell development and repertoire formation. Proc Natl Acad Sci U S A. 2002; 99(2):868-873. (Immunogen: Flow cytometry, Fluorescence activated cell sorting). View Reference
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Vance RE, Jamieson AM, Raulet DH. Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells. J Exp Med. 1999; 190(12):1801-1812. (Biology). View Reference
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Vance RE, Kraft JR, Altman JD, Jensen PE, Raulet DH. Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b). J Exp Med. 1998; 188(10):1841-1848. (Biology). View Reference
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