BD™ BV421 BCMA CAR Detection Reagent
Detection of BCMA CAR expression on transfected CHO cells. CHO cells were transfected with a CAR analog construct, comprised of cell-surface Anti-Human BCMA scFv, an epitope tag, and a transmembrane domain. The cells were then stained with BD™ BV421 BCMA CAR Detection Reagent (Cat. No. 572806/572619) and Alexa Fluor™ 647 epitope tag-specific antibody for 30 minutes at room temperature. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815) was added to cells right before analysis. The bivariate pseudocolor density plot shows BCMA CAR expression correlated with epitope tag-specific staining, gated on viable (7-AAD-negative) CHO cells. Flow cytometry was performed using a BD LSRFortessa Cell Analyzer System and FlowJo™ Software.
Detection of BCMA CAR expression on transduced primary Human T cells. Human T cells from a healthy donor were purified using the BD IMag™ Human T Lymphocyte Enrichment Set-DM (Cat. No. 557874). T cells were then activated with Anti-CD3/CD28 antibody-coupled beads plus purified BD Pharmingen™ Recombinant Human IL-2 protein (Cat. No. 554603) for 2 days, infected with BCMA CAR lentivirus, expanded for 7-10 days in culture with IL-2, and cryopreserved. Thawed BCMA CAR-transduced cells or mock transduced cells were stained with BD™ BV421 BCMA CAR Detection Reagent (Cat. No. 572806/572619) for 30 minutes at room temperature. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815) was added to cells right before analysis. Overlapping histograms show BCMA CAR staining with BD™ BV421 BCMA CAR Detection Reagent of either BCMA CAR-transduced cells (solid line) versus mock transduced cells (dashed line), gated on viable (7-AAD-negative) cells. Flow cytometry was performed using a BD LSRFortessa Cell Analyzer System and FlowJo™ Software.
Detection of BCMA CAR expression on transfected CHO cells. CHO cells were transfected with a CAR analog construct, comprised of cell-surface Anti-Human BCMA scFv, an epitope tag, and a transmembrane domain. The cells were then stained with BD™ BV421 BCMA CAR Detection Reagent (Cat. No. 572806/572619) and Alexa Fluor™ 647 epitope tag-specific antibody for 30 minutes at room temperature. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815) was added to cells right before analysis. The bivariate pseudocolor density plot shows BCMA CAR expression correlated with epitope tag-specific staining, gated on viable (7-AAD-negative) CHO cells. Flow cytometry was performed using a BD LSRFortessa Cell Analyzer System and FlowJo™ Software.
Detection of BCMA CAR expression on transduced primary Human T cells. Human T cells from a healthy donor were purified using the BD IMag™ Human T Lymphocyte Enrichment Set-DM (Cat. No. 557874). T cells were then activated with Anti-CD3/CD28 antibody-coupled beads plus purified BD Pharmingen™ Recombinant Human IL-2 protein (Cat. No. 554603) for 2 days, infected with BCMA CAR lentivirus, expanded for 7-10 days in culture with IL-2, and cryopreserved. Thawed BCMA CAR-transduced cells or mock transduced cells were stained with BD™ BV421 BCMA CAR Detection Reagent (Cat. No. 572806/572619) for 30 minutes at room temperature. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815) was added to cells right before analysis. Overlapping histograms show BCMA CAR staining with BD™ BV421 BCMA CAR Detection Reagent of either BCMA CAR-transduced cells (solid line) versus mock transduced cells (dashed line), gated on viable (7-AAD-negative) cells. Flow cytometry was performed using a BD LSRFortessa Cell Analyzer System and FlowJo™ Software.
Preparation And Storage
Recommended Assay Procedures
Surface Staining Protocol
• Resuspend up to 1 × 10^6 cells in 100 µl of stain buffer.
• Add 5 µl of the BCMA CAR Detection Reagent and incubate at either 4°C or at RT for 30 mins protected from light.
• Wash cells with stain buffer, add viability dye and proceed with acquisition.
Notes:
• Recommend using BD Pharmingen™ Human BD Fc Block™ (Cat. No. 564220) prior to staining as needed.
• For lysed whole blood staining with BD™ BV421 BCMA CAR Detection Reagent, recommend using BD™ Horizon Brilliant Stain Buffer prior to staining.
• This CAR Detection Reagent has been tested for compatibility with either BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Perm/Wash™ Buffer (Cat. No. 554723) or the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574).
Bead-based compensation or unmixing controls, such as BD™ SpectraComp™, can be used as surrogates to assess fluorescence spillover when bound to fluorochrome-conjugated antibodies or IgG Fc-fusion proteins. Although these beads have spectral properties similar to cells, variations in spectral emission may occur, resulting in differing spillover values compared to biological controls. Therefore, it is considered best practice to compare the spillover obtained from cells and bead-based controls when using BD™ SpectraComp™ for the first time, to ensure they are appropriate for the intended application.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime BD Horizon Brilliant dyes are used in a multicolor flow cytometry panel. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. When BD Horizon Brilliant Stain Buffer is used in the multicolor panel, it should also be used in the corresponding compensation controls for all dyes to achieve the most accurate compensation. For the most accurate compensation, compensation controls created with either cells or beads should be exposed to BD Horizon Brilliant Stain Buffer for the same length of time as the corresponding multicolor panel. 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
- 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).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- For U.S. patents that may apply, see bd.com/patents.
Companion Products
Chimeric Antigen Receptors (CAR) are engineered transmembrane proteins containing an extracellular single-chain domain from a proven antibody fused with major intracellular signaling components from the T Cell Receptor (TCR) complex. A CAR thus combines a defined antibody specificity with the functional capabilities of an effector cell and is a popular approach for cellular immunotherapy of cancer or autoimmunity. In CAR T cell therapy, T cells are isolated from a patient's blood, activated, transduced with a CAR gene, expanded, and reinfused into the patient, where they proliferate upon encounter with target antigen and eliminate antigen-expressing cells. Flow cytometry is used throughout this workflow to assess CAR expression and CAR-T phenotype pre-infusion, and post-infusion to probe expansion, persistence, and in vivo CAR-T characteristics. BCMA CAR Detection Reagent is a recombinant Human BCMA (aa 1-54, extracellular domain)-Mouse IgG2a Fc fusion protein conjugated to a fluorochrome that enables 1-step staining, alone or in multicolor panels, for flow cytometric analysis of BMCA CAR-expressing T, NK, or other cells.
Development References (5)
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Ali SA, Shi V, Maric I, et al. T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma.. Blood. 2016; 128(13):1688-700. (Biology). View Reference
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Hansen DK, Peres LC, Dima D, et al. Comparison of Standard-of-Care Idecabtagene Vicleucel and Ciltacabtagene Autoleucel in Relapsed/Refractory Multiple Myeloma.. J Clin Oncol. 2025; 43(13):1597-1609. (Biology). View Reference
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Hu Y, Huang J. The Chimeric Antigen Receptor Detection Toolkit.. Front Immunol. 2020; 11:1770. (Biology). View Reference
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Scheller L, Tebuka E, Rambau PF, et al. BCMA CAR-T cells in multiple myeloma-ready for take-off?. Leuk Lymphoma. 2024; 65(2):143-157. (Biology). View Reference
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Suarez ER, Chang de K, Sun J, et al. Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model.. Oncotarget. 2016; 7(23):34341-55. (Biology). View Reference
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