Igm buv395 g20 127

Manufactured by BD

IgM-BUV395 (G20-127) is a laboratory product used for flow cytometry analysis. It is an antibody labeled with the BUV395 fluorescent dye, which is designed to detect and quantify IgM proteins in biological samples.

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Lab products found in correlation

Cd19 ecd j3 119, by Beckman Coulter (3 mentions) Cd21 buv737 b ly4, by BD (2 mentions) Aqua viability dye, by Thermo Fisher Scientific (2 mentions) Fortessa, by BD (1 mentions) Cd20alexa700, by BD (1 mentions) Cd10 bv510 hi10a, by BioLegend (1 mentions) Cd27 bv605 o323, by BioLegend (1 mentions) Igg bv786 g18 145, by BD (1 mentions) Igd cy7pe, by BD (1 mentions) Recombinant bir a, by Avidity (1 mentions) Lightning link apc conjugation kit, by Abcam (1 mentions) Cd21 buv737, by BD (1 mentions) Streptavidin pe, by Thermo Fisher Scientific (1 mentions) Lsrfortessa, by BD (1 mentions) Aria 2, by BD (1 mentions)

3 protocols using igm buv395 g20 127

Identification of Influenza-Specific B Cells

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HA-specific B cells were identified within a mean of 5.6 × 10⁶ cryopreserved PBMC (range 3.6–10.9 × 10⁶ PBMC/stain) by co-staining with HA probes conjugated to SA-PE or -APC (Thermofisher)34 (link)36 (link) and SA-BB515 probe control. Monoclonal antibodies for surface staining included: CD19-ECD (J3-119) (Beckman Coulter), IgM-BUV395 (G20-127), CD21-BUV737 (B-ly4), IgD-Cy7PE (IA6-2), IgG-BV786 (G18-145) (BD), IgA-Vio450 (IS11-8E10) (Miltenyi), CD14-BV510 (M5E2), CD3-BV510 (OKT3), CD8a-BV510 (RPA-T8), CD16-BV510 (3G8), CD10-BV510 (HI10a), CD27-BV605 (O323) (Biolegend). Cell viability was assessed using Aqua Live/Dead amine-reactive dye (Thermofisher). An average of 1.38 × 10⁵ live CD19+ B cells (interquartile range 0.86–1.83 × 10⁵) were collected per subject per time point (155 samples in total) using a BD Fortessa configured to detect 18 fluorochromes and analysis was performed using FlowJo software version 9.5.2 (TreeStar).

Wheatley A.K., Kristensen A.B., Lay W.N, & Kent S.J. (2016). HIV-dependent depletion of influenza-specific memory B cells impacts B cell responsiveness to seasonal influenza immunisation. Scientific Reports, 6, 26478.

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Identification of SARS-CoV-2 S-specific B Cells

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Fluorescent B cell probes for identification of SARS-CoV-2 S-specific B cells within cryopreserved human PBMC were generated as described (Juno et al., 2020a (link)). Cells were stained with Aqua viability dye (ThermoFisher) before incubation with B cell probes (Hexapro spike or RBD) and monoclonal antibodies for surface staining CD19-ECD (J3-119) (Beckman Coulter), CD20 Alexa700 (2H7), IgM-BUV395 (G20-127), CD21-BUV737 (B-ly4), IgD-Cy7PE (IA6-2), IgG-BV786 (G18-145) (BD), CD14-BV510 (M5E2), CD3-BV510 (OKT3), CD8a-BV510 (RPA-T8), CD16-BV510 (3G8), CD10-BV510 (HI10a) (Biolegend). Single antigen-specific class-switched B cells (S or RBD⁺, CD19⁺ IgD- IgG⁺) were sorted using a BD Aria II into 96-well plates, subject to cDNA generation and multiplex PCR and Sanger sequencing, as previously described (Juno et al., 2020a (link); Tiller et al., 2008 (link)). Productive, recombined heavy (V-D-J) and light (V-J) chain immunoglobulin sequences were synthesized (Geneart) and cloned into human IgG1 expression vectors for recombinant production in Expi293 mammalian cell culture using transient transfection. After 4–5 days, IgG1 was purified from culture supernatants using Protein-A affinity chromatography.

Wheatley A.K., Pymm P., Esterbauer R., Dietrich M.H., Lee W.S., Drew D., Kelly H.G., Chan L.J., Mordant F.L., Black K.A., Adair A., Tan H.X., Juno J.A., Wragg K.M., Amarasena T., Lopez E., Selva K.J., Haycroft E.R., Cooney J.P., Venugopal H., Tan L.L., O Neill M.T., Allison C.C., Cromer D., Davenport M.P., Bowen R.A., Chung A.W., Pellegrini M., Liddament M.T., Glukhova A., Subbarao K., Kent S.J, & Tham W.H. (2021). Landscape of human antibody recognition of the SARS-CoV-2 receptor binding domain. Cell Reports, 37(2), 109822.

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Workflow for SARS-CoV-2 S-specific B Cell Profiling

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Probes for delineating SARS-CoV-2 S-specific B cells within cryopreserved human PBMC were generated by sequential addition of streptavidin-PE (Thermofisher) to trimeric S protein biotinylated using recombinant Bir-A (Avidity). SARS-CoV-2 RBD protein was directly labelled to APC using an APC Conjugation Lightning-link kit (Abcam). Cells were stained with Aqua viability dye (Thermofisher). Monoclonal antibodies for surface staining included: CD19-ECD (J3-119) (Beckman Coulter), CD20 Alexa700 (2H7), IgM-BUV395 (G20-127), CD21-BUV737 (B-ly4), IgD-Cy7PE (IA6-2), IgG-BV786 (G18-145) (BD), CD14-BV510 (M5E2), CD3-BV510 (OKT3), CD8a-BV510 (RPA-T8), CD16-BV510 (3G8), CD10-BV510 (HI10a), CD27-BV605 (O323) (Biolegend), IgA-Vio450 (clone) (Miltenyi). Cells were washed, fixed with 1% formaldehyde (Polysciences) and acquired on a BD LSR Fortessa or BD Aria II. Gating is shown in Supplementary Fig. 4.

Wheatley A.K., Juno J.A., Wang J.J., Selva K.J., Reynaldi A., Tan H.X., Lee W.S., Wragg K.M., Kelly H.G., Esterbauer R., Davis S.K., Kent H.E., Mordant F.L., Schlub T.E., Gordon D.L., Khoury D.S., Subbarao K., Cromer D., Gordon T.P., Chung A.W., Davenport M.P, & Kent S.J. (2021). Evolution of immune responses to SARS-CoV-2 in mild-moderate COVID-19. Nature Communications, 12, 1162.

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