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Two-color flow cytometric analysis of CD45RA expression on human peripheral blood lymphocytes. Human whole blood was stained with BD Horizon™ BUV563 Mouse Anti-Human CD45RA (Cat. No. 612926/612927) and APC Mouse Anti-Human CD45RO (Cat. No.559865/560899) antibodies. The erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). The two-color flow cytometric contour plot showing the correlated expression of CD45RA versus CD45RO was derived from gated events with the forward and side-light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
BD Horizon™ BUV563 Mouse Anti-Human CD45RA
Regulatory Status Legend
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. This will ensure that BD CompBeads are appropriate for your specific cellular application.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
Note: When using high concentrations of antibody, background binding of this dye to erythroid cell subsets (mature erythrocytes and precursors) has been observed. For researchers studying these cell populations, or in cases where light scatter gating does not adequately exclude these cells from the analysis, this background may be an important factor to consider when selecting reagents for panel(s).
Product Notices
- 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).
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
- BD Horizon Brilliant Ultraviolet 563 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,575,303; 8,354,239.
- CF™ is a trademark of Biotium, Inc.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
The HI100 monoclonal antibody specifically binds to the 220 kDa isoform of the human leukocyte common antigen, CD45RA. CD45RA is expressed on approximately 40-50% of peripheral CD4+ T cells, 50% of peripheral CD8+ T cells and on a portion of B cells and monocytes. The CD45RA antigen is expressed by naïve and activated T cells. CD45RA-specific antibodies are useful for the study of the suppressor/inducer subpopulation of CD4+ lymphocytes.
The antibody was conjugated to BD Horizon BUV563 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is a tandem fluorochrome of BD Horizon BUV395 which has an Ex Max of 348 nm and an acceptor dye. The tandem has an Em Max at 563 nm. BD Horizon BUV563 can be excited by the 355 nm ultraviolet laser. On instruments with a 561 nm Yellow-Green laser, the recommended bandpass filter is 585/15 nm with a 535 nm long pass to minimize laser light leakage. When BD Horizon BUV563 is used with an instrument that does not have a 561 nm laser, a 560/40 nm filter with a 535 nm long pass may be more optimal. Due to the excitation and emission characteristics of the acceptor dye, there may be spillover into the PE and PE-CF594 detectors. However, the spillover can be corrected through compensation as with any other dye combination.
Development References (3)
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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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Koristka S, Cartellieri M, Theil A, et al. Retargeting of human regulatory T cells by single-chain bispecific antibodies.. J Immunol. 2012; 188(3):1551-8. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
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Zola H. Leukocyte and stromal cell molecules : the CD markers. Hoboken, N.J.: Wiley-Liss; 2007.
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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.