Ramos B cell calcium flux was measured as described (75 (link)). Protein tetramers were formed at a 4:1 molar ratio of protein to streptavidin (Invitrogen). Ramos cell lines stably expressing DH270 UCA, DH270.1, or CH65 IgM (76 (link)) were passaged (1:10) 4 days before calcium flux experiments. On the day of the experiment, cells with >95% viability were resuspended at 106 cells ml−1 in 2:1 ratio of RPMI media (GIBCO) + FLIPR Calcium 6 dye (Molecular Devices). Cells were plated in a U-bottom 96-well tissue culture plate (Costar) and incubated at 37°C 5% CO2 for 2 hours. In a black clear-bottom 96-well plate (Costar) containing 50 μl of RPMI media (GIBCO) + FLIPR Calcium 6 dye (Molecular Devices) (2:1 ratio) either 0.1 nmol of proteins or 50 μg ml−1 of anti-human IgM F(ab’)2 (Jackson Immuno) were added (based on a 100-μl volume). Using a FlexStation 3 multimode microplate reader (Molecular Devices), 50 μl of supernatant containing cells were transferred into the 50 μl of media containing protein or anti-human IgM F(ab’)2 (Jackson Immuno) and continuously read for 5 min. Relative fluorescent value units were background-subtracted and the data expressed as percentage of the IgM maximum signal (% IgM max).
Anti human igm f ab 2
Manufactured by Jackson ImmunoResearch
Sourced in United States
Anti-human IgM F(ab')2 is a laboratory reagent produced by Jackson ImmunoResearch. It is a fragment of an antibody that specifically binds to human IgM antibodies.
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Lab products found in correlation
Anti cd19 magnetic beads, by Miltenyi Biotec (3 mentions)
Anti human ig a g m f ab 2, by Jackson ImmunoResearch (3 mentions)
Rhil 2, by Thermo Fisher Scientific (2 mentions)
Anti cd3, by BioLegend (2 mentions)
Cd40l, by Enzo Life Sciences (2 mentions)
Rhil 10, by Miltenyi Biotec (2 mentions)
Gardiquimod, by InvivoGen (2 mentions)
Cpg odn 2006, by InvivoGen (2 mentions)
Rpmi media, by Thermo Fisher Scientific (1 mentions)
Flexstation 3 multi mode microplate reader, by Molecular Devices (1 mentions)
Flipr calcium 6 dye, by Molecular Devices (1 mentions)
Anti mouse igm f ab 2, by Jackson ImmunoResearch (1 mentions)
Protease and phosphatase inhibitor cocktail, by Nacalai Tesque (1 mentions)
Anti cd43 magnetic beads, by Miltenyi Biotec (1 mentions)
Syk d3z1e, by Cell Signaling Technology (1 mentions)
Foxp3 transcription factor staining buffer kit, by Thermo Fisher Scientific (1 mentions)
Live dead cell assay, by Thermo Fisher Scientific (1 mentions)
Fc block, by BioLegend (1 mentions)
18 color lsr 2 flow cytometer, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
10 protocols using anti human igm f ab 2
1
Ramos B cell calcium flux assay
2
B Cell Activation and Analysis
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Freshly isolated or thawed PBMC were washed with complete medium and counted. For qPCR, B cells were positively selected using anti-CD19 magnetic beads (Miltenyi Biotech). 0.5–1.0 × 106 cells per well were seeded in round-bottom 96-well plates in 200μL 10% FBS/RPMI 1640. Cells were stimulated with anti-human IgM F(ab′2) or anti-human Ig(A + G + M) F(ab′2) (Jackson Immuno-Research Laboratories) for indicated times. After stimulation, cells were collected for flow cytometry or qPCR.
3
Activated B-cell Proliferation Assay
4
B Cell Activation and Analysis
5
Activation and Viability of Splenic B Cells
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Splenic B cells were purified by negative selection of CD43+ cells using anti-CD43 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The purified B cells (1 × 107 cells/ml) were stimulated with 10 µg/mL anti-mouse IgM F(ab)′2 (Jackson ImmunoResearch, West Grove, PA, USA) or 10 µg/mL anti-human IgM F(ab)′2 (Jackson ImmunoResearch) and then lysed in lysis buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100 and 0.5 mM EDTA plus protease and phosphatase inhibitor cocktails (Nacalai Tesque)]. Samples were transferred to polyvinylidene difluoride membranes by electrophoresis and analysed by immunoblotting with antibodies against p-Syk (Tyr525/526) (C87C1) and Syk (D3Z1E) (Cell Signaling Technology, Danvers, MA, USA). The purified splenic B cells were cultured with or without 25 ng/ml BAFF (R&D Systems, Minneapolis, MN, USA) for 24, 48, or 72 h. The frequency of live B cells was assessed using TOPRO3 (Invitrogen) exclusion.
6
B-cell Activation Assay Using T-cell Supernatant
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Purified CD19+ lymphocytes (1 × 105 cells/well) were cultured in 96‐well plates in 50 μL medium (RPMI, 5%FBS) supplemented with 50 ng/mL rhIL‐10 (Miltenyi Biotec), 20 U rhIL‐2 (PeproTech), 0.1 μg/mL CD40L (Enzo Life Science) and 25 μg/mL anti‐human IgM F(ab’)2 (Jackson ImmunoResearch). Purified CD4+ lymphocytes (2.5 × 105‐3.5 × 105 cells/well) were cultured for 36 h with rhIL‐2 (PeproTech, 50U/mL) at 37°C, 5% CO2 in 48‐well plates that had been coated overnight with anti‐CD3 (2 μg/mL) (BioLegend, clone OKT3) +/‐anti‐CD46 (2 μg/mL) (R&D Systems, clone 344519). To assess B‐cell activation, 100 μL of T‐cell supernatant was added to B cells and cultured for 5–10 days at 37°C, 5%CO2. To block the activity of IL‐10, a neutralizing mAb to IL‐10 (2.5 μg/mL) was used. To restore B‐cell proliferation in mixtures containing T‐cell supernatants rhIL‐10 (1 μg/mL) was added.
7
PBMC Isolation, Stimulation, and Flow Cytometry
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Freshly isolated or thawed PBMC were washed with complete medium and counted. For qPCR, B cells were positively selected using anti-CD19 magnetic beads (Miltenyi Biotech). 0.5-1.0x10 6 cells per well were seeded in round-bottom 96-well plates in 200µl 10%FBS/RPMI 1640. Cells were stimulated with anti-human IgM F(ab¢2) or anti-human Ig(A+G+M) F(ab¢2) (Jackson Immuno-Research Laboratories) for indicated times. After stimulation, cells were collected for flow cytometry or qPCR.
To stain for flow cytometry, PBMC or tonsil cells were first incubated with Fc-Block (Biolegend) for 5 minutes at RT. Then 100ul of surface antibody cocktail (table S4) was added to each tube and incubated on ice for 30 minutes. Next, live/dead cell assay (Invitrogen) was applied for 15 minutes on ice. Intracellular staining was performed using Foxp3/Transcription factor staining buffer kit (eBioscience) following manufacture protocol. After intracellular staining, cells were washed and fixed in 100µl PBS/1% paraformaldehyde for 20 minutes at RT and then 200µl of PBS/5%BSA was added to dilute the fixative. Samples were analyzed on an 18-color LSR II flow cytometer (eBioscience) on the same day or after kept at 4°C overnight. Analyses were performed using FlowJo v.10 (Tree Star) and doublet exclusion was performed on all samples. Gating strategy is depicted in fig. S8.
To stain for flow cytometry, PBMC or tonsil cells were first incubated with Fc-Block (Biolegend) for 5 minutes at RT. Then 100ul of surface antibody cocktail (table S4) was added to each tube and incubated on ice for 30 minutes. Next, live/dead cell assay (Invitrogen) was applied for 15 minutes on ice. Intracellular staining was performed using Foxp3/Transcription factor staining buffer kit (eBioscience) following manufacture protocol. After intracellular staining, cells were washed and fixed in 100µl PBS/1% paraformaldehyde for 20 minutes at RT and then 200µl of PBS/5%BSA was added to dilute the fixative. Samples were analyzed on an 18-color LSR II flow cytometer (eBioscience) on the same day or after kept at 4°C overnight. Analyses were performed using FlowJo v.10 (Tree Star) and doublet exclusion was performed on all samples. Gating strategy is depicted in fig. S8.
8
B Cell Gene Expression and Proliferation
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B cells were cultured in R10 medium at 37 °C and 5% CO2. For gene expression analyses, cells were enriched for CD27–CD10– B cells and plated at 3 × 106 cells per ml with preincubation for 1 hour at 37 °C with Emab (5 μg/ml) or hIgG1 isotype control (5 μg/ml; Sigma) or R10 medium and then stimulated with TLR7 agonist R848 (50 ng/ml; InvivoGen), F(ab′)2 anti-human IgM (5 μg/ml; Jackson ImmunoResearch Laboratories, Inc.), or a combination of R848 plus anti-IgM. In some cases, cells were pretreated with IFN-α at 1000 U/ml (PBL, Piscataway, NJ, USA). Samples were cultured for 12 hours, harvested, and used for RNA isolation. For cell proliferation experiments, cells were loaded with 2.5 μM CFSE (ThermoFisher Scientific) in PBS at 37 °C for 10 min, quenched with R10 medium, washed, stimulated, and then cultured for 3 days. For assessing cell survival and plasma cell differentiation, sorted B-cell subsets were placed in 96-well plates at a concentration of 2.5 × 106 cells per ml, treated with different stimuli, and analyzed by flow cytometry after 3–5 days of in-vitro cell culture.
9
B Cell Activation and Differentiation
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Total CD20+ B cells were plated at 100,000 cells/well in a 96-well-plate in RPMI 10% FBS and 2.5 μg/ml polyclonal F(ab’)2 anti-human IgM (Jackson Immunoresearch), 1.0 μg/ml CpG ODN2006 (In vivogen) or 2 μg/ml Gardiquimod (In vivogen). Expression of surface activation markers CD69 and CD86 was analyzed on gated CD19+CD27- IgD+ or IgD- cells after 48 hours by flow cytometry. For functional analysis of IGHD variants flow cytometrically sorted CD19+CD27IgM+IgD+ and CD19+CD27-IgM+IgD- B cells were stimulated by anti-human IgM or anti-human IgD antibodies. Expression of CD69 was analyzed after 48 hours.
10
Activation of Human B Cells
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