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Two-color flow cytometric analysis of CD28 expression on mouse splenocytes. BALB/c mouse spleen cells were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with APC Hamster Anti-Mouse CD3e antibody (Cat. No. 553066/561826) and either BD Horizon™ BV421 Mouse IgG2b, κ Isotype Control (Cat. No. 562748; Left Plot) or BD Horizon BV421 Mouse Anti-CD28 antibody (Cat. No. 566816; Right Plot) at 0.5 μg/test. A two-color flow cytometric dot plot showing the correlated expression of CD3e versus CD28 (or Ig Isotype control staining) was derived from gated events with the forward and side light-scatter characteristics of viable leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
BD Pharmingen™ BV421 Mouse Anti-Mouse CD28
Regulatory Statusの凡例
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
推奨アッセイ手順
BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome-conjugated antibodies are bound to 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 CompBead to ensure that BD Comp beads 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).
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
- BD Horizon Brilliant Violet 421 is covered by one or more of the following US patents: 8,158,444; 8,362,193; 8,575,303; 8,354,239.
- 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.
- 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.
関連製品
The E18 monoclonal antibody specifically recognizes CD28 that is encoded by Cd28 (CD28 antigen) which belongs to the immunoglobulin gene superfamily. CD28 is a ~45 kDa type I transmembrane glycoprotein that can be expressed as a disulfide-linked homodimer. The extracellular region of CD28 contains an IgV-like domain which is followed by a transmembrane region and a cytoplasmic domain. CD28 is expressed on thymocytes, most peripheral CD4+ T cells and CD8+ T cells, NK cells, and plasmacytoid dendritic cells. CD28 functions as a costimulatory receptor upon binding ligands that include CD80 (B7-1) and CD86 (B7-2) that are expressed on antigen presenting cells and B cells. CD28-mediated costimulation augments the proliferation and cytokine production of activated T and NK cells, provides a costimulatory signal for CTL induction, and promotes T cell survival. There is considerable evidence that CD28 is a costimulatory receptor involved in many, but not all, T cell-dependent immune responses. The E18 antibody reportedly blocks the binding of B7 antigens to CD28 and can costimulate T cell activation and proliferation. Spleen cells from a CD28-deficient mouse that was immunized with mouse CD28 were used to generate the hybridoma that secretes the E18 antibody.
The antibody was conjugated to BD Horizon BV421 which is part of the BD Horizon Brilliant™ Violet family of dyes. With an Ex Max near 407 nm and Em Max near 421 nm, BD Horizon BV421 can be excited by the violet laser (405 nm) and detected with a 450/50 nm filter. BD Horizon BV421 conjugates are very bright, often exhibiting a 10-fold improvement in brightness compared to Pacific Blue™ conjugates. Due to nearly identical excitation and emission properties but different spillover characteristics, BD Horizon BV421, Pacific Blue, and BD Horizon V450 cannot be used simultaneously.
Development References (3)
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Beyersdorf N, Ding X, Blank G, Dennehy KM, Kerkau T, Hünig T. Protection from graft-versus-host disease with a novel B7 binding site-specific mouse anti-mouse CD28 monoclonal antibody.. Blood. 2008; 112(10):4328-36. (Clone-specific: Blocking, Functional assay). View Reference
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Dennehy KM, Elias F, Zeder-Lutz G, et al. Cutting edge: monovalency of CD28 maintains the antigen dependence of T cell costimulatory responses.. J Immunol. 2006; 176(10):5725-9. (Immunogen: Flow cytometry). View Reference
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Macal M, Tam MA, Hesser C, et al. CD28 Deficiency Enhances Type I IFN Production by Murine Plasmacytoid Dendritic Cells. J Immunol. 2016; 196(4):1900-1909. (Clone-specific: Flow cytometry). View Reference
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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.