PBMCs or B cells were stained for 20 min with fluorescent conjugates of CD19-Alexa Fluor 700, CD38-PerCP.Cy5.5, CD39-FITC and CD73-PE (Biolegend, San Diego, CA) IgD-BV421, IgM-BV605 (BD Biosciences, San Jose, CA) CD27−APC and CD24-APC eFluor780 (eBioscience, San Diego, CA), and a viability marker (LIVE/DEADTM Fixable aqua dead cell stain, ThermoFisher Scientific, Waltham, MA). Cells were washed (centrifuged for 5 min 300 × g at room temperature) and resuspended in PBS and acquired within 24 h on a BD LSR FortessaTMX-20. Compensation beads (BD, Biosciences, San Jose, CA) were used to optimize fluorescence compensation settings for multicolour flow cytometric analysis. A minimum of 100,000 events in the lymphocyte gate was collected. Naïve and Memory B cell subsets were defined as in our previous publication (16 (link)) based on the classification described in Ref (20 (link)). Representative plots of the classical B-cell subsets defined by IgD/CD27 and IgD/CD38 using this system are shown in Supplementary Figure 1 .
Igm bv605
Manufactured by BD
Sourced in United States, Germany
IgM-BV605 is a fluorochrome-conjugated antibody that binds to the IgM protein. It is designed for use in flow cytometry applications to identify and quantify IgM-positive cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Compensation beads, by BD (2 mentions)
Lsrfortessatm x 20, by BD (2 mentions)
Igd bv421, by BD (2 mentions)
Cd27 apc and cd24 apc efluor780, by Thermo Fisher Scientific (2 mentions)
Igd bv711, by BD (2 mentions)
Brilliant staining buffer, by BD (2 mentions)
Cd39 fitc, by BioLegend (1 mentions)
Cd73 pe, by BioLegend (1 mentions)
Cd38 percp cy5, by BioLegend (1 mentions)
Cd19 alexa fluor 700, by BioLegend (1 mentions)
Live deadtm fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Cd19 alexa fluor 700, by BD (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)
Cd8 buv496, by BD (1 mentions)
Phorbol myristate acetate (pma), by Thermo Fisher Scientific (1 mentions)
Live dead stain, by Thermo Fisher Scientific (1 mentions)
Cd20 apc cy7, by BD (1 mentions)
Cd3 bv650, by BD (1 mentions)
Golgistop, by BD (1 mentions)
7 protocols using igm bv605
1
Multiparametric flow cytometry of B cell subsets
2
Comprehensive B Cell Immunophenotyping
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Flow cytometry was used to follow changes in classical B cell immunophenotype markers (CD19, IgD, IgM, and CD27) as well as those involved in energy pathways including CD24, CD39, CD73, and CD38 throughout in vitro culture. B cell numbers, B cell growth (total mass), and viability were also calculated using flow cytometry. Briefly, PBMCs or B cells were stained for 20 min with fluorescent conjugates of monoclonal antibodies to CD19-Alexa Fluor 700, IgD-BV421, and IgM-BV605 (BD Biosciences, San Jose, CA, USA), CD27-APC and CD24-APC eFluor780 (eBioscience, San Diego, CA, USA), CD39-FITC, CD73-PE, and CD38-PerCP.Cy5.5 (Biolegend, San Diego, CA, USA), and a viability marker (LIVE/DEAD™ Fixable aqua dead cell stain, ThermoFisher Scientific, Waltham, MA, USA). The cells were washed and resuspended in PBS and acquired within 24 h on a BD LSR FortessaTMX-20. Total mass was calculated by combining the mass of debris, dead cells, and live cells in each culture well using the following formula: (FSC × mean number viable + FSC × mean number dead + FSC × mean number debris). Compensation beads (BD, Biosciences, San Jose, CA, USA) were used to optimize the fluorescence compensation settings for multicolor flow cytometric analysis. A minimum of 100,000 events in the lymphocyte gate was collected.
3
Quantifying IL-9 and IL-33 T Cells in Rhesus PBMCs
4
Single B-cell Sorting for MPER Analysis
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Fluorescent-labeled antibodies CD3-Alexa Fluor 700 (BD 557943), CD8-Alexa Fluor 700 (BD 561453), CD14-PE-Cy7 (BD 561385), CD16-PE-Cy7 (BD 560716), CD19 PerCP*Cy5.5 (BD 561295), CD20 PerCP*Cy5.5 (BD 350955), IgG-PE-Cy5 (BD 551497), IgM-BV605 (BD 562977), and IgD-BV605 (BD 563313), which target cell surface markers, were used as 1:200-fold dilution in staining. Biotin-labeled MPER peptide PDT-081 (E654KNEQELLELDKWASLWNWFDITNWLWYIK683-biotin) was purchased from GenScript and coupled separately to streptavidin-BV421 (BD 562426) and streptavidin-APC (allophycocyanin, BD 555335). PBMCs were stained using the LIVE/DEAD Fixable Near-IR Dead Cell Kit (Life Technologies, L34957) for 30 min on ice. Cells were then labeled with antibodies cocktail along with MPER probes for 1 h in Brilliant Staining buffer (BD 563794) on ice. Cell population CD19+/CD20+, CD3−/CD8−, CD14−/CD16−, IgG+, IgD-/IgM− MPER double positive were sorted using BD FACSAria III sorter into individual wells of a 96-well plate containing lysis buffer and plates were immediately sealed and stored at −80 °C. The single B-cell sorting strategy is shown in the Supplementary Fig. 9 .
5
Immunophenotyping Immortalized B Cells
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Mock- or MCM-treated immortalized B cells or TAB were stained with the following antibodies or matched isotypes and analyzed on a FACS Aria III (BD): CD19 BV711 (clone SJ25C1, 0.06 µg/100 µl, catalog number 563036), CD20 AF700 (clone 2H7, 0.5 µg/100 µl, 560631), CD24 PE-CF594 (clone ML5, 1 µg/100 µl, 562405), CD27 BV421 (clone M-T271, 0.25 µg/100 µl, 562513), CD38 APC (clone HIT2, 0.125 µg/100 µl, 555462), CD138 PE (clone MI15, 0.125 µg/100 µl, 552026), IgD PE-Cy7 (clone IA6-2, 0.125 µg/100 µl, 561314), IgG FITC (clone G18-145, 0.125 µg/100 µl, 555786), IgM BV605 (clone G20-127, 0.5 µg/100 µl, 562977) (all BD biosciences). Live/dead cell exclusion was performed by addition of 7-AAD (5 µg/ml, Calbiochem) prior to acquisition of the samples. Data were analyzed using FlowJo 10.4.2 (FlowJo LLC). The gating strategy is shown in Supplementary Fig. 10 .
6
Flow Cytometry Analysis of Splenocytes
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Mice that were immunized as described above were sacrificed 4 weeks after the third immunization. Spleens were collected into RPMI containing 2% fetal bovine serum and manually homogenized using the back of a syringe plunger. Cells were filtered through 75 um mesh, washed 1x, and counted. 2x107 splenocytes were stained for flow cytometry. All washes for the staining process were performed in PBS containing 2% fetal bovine serum and 2 mM EDTA. Cells were incubated with CD16/32 (Biolegend Cat# 101302) and 5.875 μg/ml of biotinylated Abp2DRBD for 10 minutes, then washed 3x. A cocktail containing the following antibodies was prepared in BD Brilliant Staining Buffer (BD Cat. # 563794), all sourced from BioLegend unless otherwise indicated : Zombie NIR (Cat#423105), CD19-BV750 (Cat#115561), CD4-BV570 (Cat#100542), IgD-BV711 (Cat#405731), IgM-BV605 (Cat#406523), IgG1-BV510 (Cat#406621), Fas-PE (BD Cat# 554258), GL7-PcpCy5.5 (Cat# 144610), CD38-PE-Cy7 (Cat#102718), CD138-BV421 (Cat#142508), and streptavidin-APC-Fire-750 (Cat#405250). Invitrogen UltraComp eBeads were used for single colors. Flow cytometry data was collected using the Cytek Aurora with 4 laser 16V-14B-10YG-8R configuration and processed on FlowJo10 for Mac.
7
Comprehensive Immunological Assays for In-Depth Analysis
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Gum arabic, activated carbon, 2,7-dichlorofluorescein diacetate (DCFDA), toluidine blue, protease inhibitor cocktail tablets, phosphatase inhibitor cocktail tablets, MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel were purchased from Merck KGaA (Darmstadt, Germany). 25% Aqueous Solution Glutaraldehyde, Paraformaldehyde, Sorensen's Phosphate Buffer, 2% Aqueous Solution Osmium Tetroxide, Ethyl Alcohol, Acetone, Araldite, Dibutyl phthalate (DBP) were purchased Electron Microscopy Sciences (Hatfield, USA). Leukocyte Activation Cocktail with BD GolgiPlug™, FACS antibodies include anti-mouse I-A/I-E-BV510, IgG1-BB700, IgM-BV605, IgE-BV786, IgD-BV711, CD1d-BV421, CD5-PE, CD45-BUV395, CD19-APC, CD45R/B220-BUV496, CD45-APC-Cy7, CD3e-FITC, CD4-V450, CD8-BV510, CD25-BV605, IL-4-PE-Cy7, IFN-γ-PE, FoxP3-AF647, CD103-BUV395, F4/80-BV711, CD80-BV650, CD11b-BV510, Ly6-G-PerCP Cy5.5, PE-Ly6-C, CD45-APC-Cy7, CD11c--AF700 were purchased from BD (Heidelberg, Germany); Another FACS antibody Anti-mouse-IL-17A-BV650 was purchased from eBioscience (Frankfurt am Main, Germany). Anti-mouse HMGB1, Anti-mouse phospho-p65, anti-mouse phospho-p38, anti-mouse GAPDH and anti-rabbit IgG HRP were obtained from Cell signaling Technology (Frankfurt am Main, Germany). IgE and histamine ELISA kits were purchased from Abcam (Berlin, Germany).
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