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RY586 Mouse Anti-Human CD28
RY586 Mouse Anti-Human CD28
Multiparameter flow cytometric analysis of CD28 expression on Human peripheral blood leucocyte populations. Human whole blood was stained with either BD Horizon™ RY586 Mouse IgG1, κ Isotype Control (Cat. No. 568097; Left Plot) or BD Horizon™ RY586 Mouse Anti-Human CD28 antibody (Cat. No. 568731/568732; Right Plot). Erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). The bivariate pseudocolor density plot showing CD28 expression (or Ig Isotype control staining) versus side light-scatter (SSC-A) signals was derived from events with the forward and side light-scatter characteristics of intact leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Multiparameter flow cytometric analysis of CD28 expression on Human peripheral blood leucocyte populations. Human whole blood was stained with either BD Horizon™ RY586 Mouse IgG1, κ Isotype Control (Cat. No. 568097; Left Plot) or BD Horizon™ RY586 Mouse Anti-Human CD28 antibody (Cat. No. 568731/568732; Right Plot). Erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). The bivariate pseudocolor density plot showing CD28 expression (or Ig Isotype control staining) versus side light-scatter (SSC-A) signals was derived from events with the forward and side light-scatter characteristics of intact leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Product Details
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BD Horizon™
CD28 antigen; T44; Tp44; TP44
Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Mouse C3H x BALB/c IgG1, κ
Human CD28 Transfected Cell Line
Flow cytometry (Routinely Tested)
5 µl
V 5T CD28.05
940
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

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 and unreacted dye was removed.

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

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. 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.
  3. 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).
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. An isotype control should be used at the same concentration as the antibody of interest.
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
  8. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  9. CF™ is a trademark of Biotium, Inc.
Antibody Details
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CD28.2

The CD28.2 monoclonal antibody specifically binds to CD28, a 44 kDa homodimeric transmembrane glycoprotein present on most mature T cells, thymocytes and plasma cells. CD28 is a costimulatory receptor that binds CD80 and CD86 as ligands and plays a very important role in T cell-B cell interactions. It has been suggested that CD28 initiates and regulates a separate and distinct signal transduction pathway from those stimulated by the TCR complex. Additionally, it has been reported that CD28 antibody clones vary in their ability to stimulate T cells to produce IL-2 and increase intracellular Ca2+ concentration. This finding suggests the existence of functionally distinct subregions on the CD28 molecule. CD28.2 has been demonstrated to bind to the same molecule as clone L293, another CD28 mAb, and has been reported to induce Ca2+ influx in Jurkat T cells.

Format Details
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RY586
The BD Horizon RealYellow™ 586 (RY586) Dye is part of the BD family of yellow-green dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 565-nm and an emission maximum (Em Max) at 586-nm. Driven by BD innovation, RY586 can be used on both spectral and conventional cytometers and is designed to be excited by the Yellow-Green laser (561-nm) with minimal excitation by the 488-nm Blue laser. For conventional instruments equipped with a Yellow-Green laser (561-nm), RY586 can be used as an alternative to PE and we recommend using an optical filter centered near 586-nm (eg, a 586/15-nm bandpass filter). For spectral instruments equipped with a Yellow-Green laser (561-nm), it can be used in conjunction with PE. Compared to PE, RY586 is similar in brightness, minimal spillover into Blue detectors, and increased spillover into the 610/20-nm (PE-CF594) detector. Please ensure that your instrument configuration (lasers and optical filters) is appropriate for this dye.
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RY586
Yellow-Green 561 nm
564 nm
586 nm
Citations & References
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Development References (7)

  1. Barclay NA, Brown MH, Birkeland ML, et al, ed. The Leukocyte Antigen FactsBook. San Diego, CA: Academic Press; 1997.
  2. June CH, Bluestone JA, Nadler LM, Thompson CB. The B7 and CD28 receptor families. Immunol Today. 1994; 15(7):321-331. (Biology). View Reference
  3. Kuiper H, Brouwer M, Vermeire S, van Lier R. Analysis of the Workshop CD28 Panel mAb: distinct signalling pathways coupled to CD28. In: Schlossman SF. Stuart F. Schlossman .. et al., ed. Leucocyte typing V : white cell differentiation antigens : proceedings of the fifth international workshop and conference held in Boston, USA, 3-7 November, 1993. Oxford: Oxford University Press; 1995:373-374.
  4. Nunes J, Klasen S, Franco MD, et al. Signalling through CD28 T-cell activation pathway involves an inositol phospholipid-specific phospholipase C activity. Biochem J. 1993; 293(3):835-842. (Clone-specific: Calcium Flux, (Co)-stimulation, Functional assay). View Reference
  5. Nunes J, Klasen S, Ragueneau M, et al. CD28 mAbs with distinct binding properties differ in their ability to induce T cell activation: analysis of early and late activation events. Int Immunol. 1993; 5(3):311-315. (Immunogen: Calcium Flux, (Co)-stimulation, Flow cytometry, Functional assay, IC/FCM Block, Immunoprecipitation, Stimulation). View Reference
  6. Olive D, Cerdan C, Costello R, et al. CD28 and CTLA-4 cluster report. In: Schlossman SF. Stuart F. Schlossman .. et al., ed. Leucocyte typing V : white cell differentiation antigens : proceedings of the fifth international workshop and conference held in Boston, USA, 3-7 November, 1993. Oxford: Oxford University Press; 1995:360-370.
  7. Thaker YR, Schneider H, Rudd CE. TCR and CD28 activate the transcription factor NF-κB in T-cells via distinct adaptor signaling complexes.. Immunol Lett. 2015; 163(1):113-9. (Clone-specific: Activation, Flow cytometry). View Reference
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