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Multicolor flow cytometric analysis of CD45RA expression on Human peripheral blood leukocytes. Human whole blood was stained with BD Horizon™ RY703 Mouse Anti-Human CD45RO (Cat. No. 571463; Bottom Plots) and with either BD Horizon™ RB670 Mouse IgG1, κ Isotype Control (Cat. No. 571784; Left Plots) or BD Horizon™ RB670 Mouse Anti-Human CD45RA antibody (Cat. No. 571965/571966; Right Plots). Erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). The bivariate pseudocolor density plots showing the correlated expression of CD45RA (or Ig Isotype control staining) versus either side light-scatter (SSC-A) signals (Top Plots) or CD45RO (Bottom Plots) were derived from gated events with the forward and side light-scatter characteristics of either viable leukocytes or lymphocytes, respectively. Samples were acquired using a BD FACSymphony™ A5 SE Cell Analyzer and spectrally unmixed using FlowJo™ v10.10 Software.
BD Horizon™ RB670 Mouse Anti-Human CD45RA
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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
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.
- 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.
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
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- 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.
- 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.
- 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.
- Clones without a listed workshop have not been investigated in an HLDA workshop to receive a CD nomenclature. We use “CD” provisionally when our internal testing indicates that this clone binds to the same CD antigen as workshopped clones.
Companion Products
The 5H9 monoclonal antibody specifically binds to the human form of the leukocyte common antigen (LCA), CD45RA. This clone also crossreacts with CD45RA expressed by peripheral blood lymphocytes, monocytes and some granulocytes from rhesus macaque, baboon, and cynomolgus monkeys. The observed frequency of CD45RA positive lymphocytes is higher in the non-human primate compared to what is seen with normal human donor lymphocytes.
Development References (6)
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Arbore G, West EE, Rahman J, et al. Complement receptor CD46 co-stimulates optimal human CD8+ T cell effector function via fatty acid metabolism.. Nat Commun. 2018; 9(1):4186. (Clone-specific: Flow cytometry). View Reference
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Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:1-1182.
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Palermo B, Franzese O, Donna CD, et al. Antigen-specificity and DTIC before peptide-vaccination differently shape immune-checkpoint expression pattern, anti-tumor functionality and TCR repertoire in melanoma patients.. Oncoimmunology. 2018; 7(12):e1465163. (Clone-specific: Flow cytometry). View Reference
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Reimann KA, Waite BC, Lee-Parritz DE, et al. Use of human leukocyte-specific monoclonal antibodies for clinically immunophenotyping lymphocytes of rhesus monkeys. Cytometry. 1994; 17(1):102-108. (Biology). View Reference
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Sopper S, Stahl-Hennig C, Demuth M, Johnston IC, Dorries R, ter Meulen V. Lymphocyte subsets and expression of differentiation markers in blood and lymphoid organs of rhesus monkeys. Cytometry. 1997; 29(4):351-362. (Biology). View Reference
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van der Windt DJ, Dons EM, Montoya CL, et al. T-lymphocyte homeostasis and function in infant baboons: implications for transplantation. Transplantation. 2012; 25(2):218-228. (Clone-specific: Flow cytometry). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.