Splenic B cells were enriched by depletion of CD43+ cells with magnetic anti-mouse-CD43 microbeads (Miltenyi Biotech Cat# 130-049-801, RRID: AB_2861373), transduced with in-house generated TAT-Cre recombinase (42 (link), 43 (link)), cultured in the absence or presence of LPS (20 μg/ml, Escherichia coli 055:B5; Sigma Cat# L2880) or F(ab’)2 fragment anti-IgM (1.2 μg/ml; Jackson ImmunoResearch Labs Cat# 115-006-020; RRID: AB_2338469) and 1 µM BrdU or cultured with LPS plus recombinant mouse IL-4 (10–20 units/ml; Peprotech Cat# 214-14).
Anti igm f ab 2 fragment
Manufactured by Jackson ImmunoResearch
Sourced in United States
Anti-IgM F(ab')2 fragment is a laboratory reagent derived from goat anti-human IgM antibodies. It is designed to be used as a secondary detection reagent in various immunoassay and immunohistochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (3 mentions)
Hrp conjugated anti rabbit or anti mouse antibodies, by Jackson ImmunoResearch (2 mentions)
Cd40l, by R&D Systems (2 mentions)
Mini protean system, by Bio-Rad (2 mentions)
Recombinant mouse il 4, by Thermo Fisher Scientific (1 mentions)
Escherichia coli 055 b5, by Merck Group (1 mentions)
F ab 2 fragment alexa fluor 594 conjugate, by Cell Signaling Technology (1 mentions)
Tx 100, by Merck Group (1 mentions)
Dylight 594 phalloidin, by Cell Signaling Technology (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
P src, by Cell Signaling Technology (1 mentions)
Pstat3 ser, by Cell Signaling Technology (1 mentions)
Cdc42, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Total erk, by Cell Signaling Technology (1 mentions)
Anti cd40, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Hrp conjugated anti rabbit or anti mouse abs, by Jackson ImmunoResearch (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
10 protocols using anti igm f ab 2 fragment
1
Splenic B Cell Purification and Stimulation
2
Imaging Activated CLL Cells
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CLL cells (20 × 106/ml) were seeded on poly-L-lysine–coated glass and then left either inactivated or activated with F(ab′)2 Fragment anti-IgM or F(ab′)2 Fragment anti-IgG at 10 µg/ml (Jackson ImmunoResearch, PA, USA) for 5, 15, and 40 min. The cells were fixed with 4% methanol-free paraformaldehyde for 20 min, followed by permeabilization with 0.2% TX-100 (Sigma Aldrich, MO, USA). The antibodies used were fluorescein isothiocyanate (FITC)-conjugated anti-IgM, FITC-conjugated anti-IgG (Bethyl, TX, USA) (1:200) for 60 min, and DyLight 594 Phalloidin (Cell Signaling Technology, MA, USA) (1:20) for 20 min. Phospho-CD79a (Tyr182) (1:200) and anti-rabbit IgG (H+L), F(ab′)2 Fragment (Alexa Fluor® 594 Conjugate) (1:500), Cell Signaling Technology (Beverly, MA), were used for pCD79a staining. LAMP1 (1:100) and anti-mouse IgG (H+L), F(ab′)2 Fragment (Alexa Fluor® 594 Conjugate) (1:500), were used for LAMP1 staining. After incubation, cells were washed three times with 1% bovine serum albumin (BSA) in PBS. All the cells were labeled with fluorescent mounting medium with DAPI (Golden Bridge International, Inc., WA, USA). Images were acquired using Leica SP8 confocal microscope (Sackler Faculty of Medicine core facility, Tel Aviv University). Quantification was performed using Imaris (version 9.3.1) software.
3
Signaling Pathway Analysis in Purified B Cells
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Purified B cells were left at 37°C for 10 min in chamber buffer (PBS, 0.5% FCS, 1 g/liter d -Glucose, 2 mM MgCl2, and 0.5 mM CaCl2) to equilibrate before stimulation. They were then stimulated for various times with 10 µg/ml anti-IgM F(ab′)2 fragment (Jackson ImmunoResearch Laboratories), 10 µg/ml anti-κ (HB558; American Type Culture Collection), or 1 µg/ml CD40L (R&D Systems).
For Cdc42-GTP pull-down experiment, a commercial kit (Cytoskeleton Inc.) was used following the manufacturer’s instructions, specifically using 107 B cells for 15 µg of beads. For immunoblotting, stimulated cells were then lysed in lysis buffer (20 mM Tris-HCL, pH 8.0,150 mM NaCl, 5 mM EDTA, Protease Inhibitor cocktail [Roche], 10 mM NaF, 1 mM Na3VO4, and 1% NP-40) for 15 min on ice, and samples were loaded into 4–20% (for Cdc42-GTP pull-down) or 12% (for all other applications) for electrophoresis. Proteins were detected with antibodies against pErk, pAkt (S473), pCD19, pSyk, pSrc, totalErk, pSTAT3 (Ser), and Cdc42 (all from Cell Signaling Technology) using the secondary HRP-conjugated anti–rabbit or anti–mouse antibodies (Jackson ImmunoResearch Laboratories).
Blot densitometry analysis was performed using the ImageJ (National Institutes of Health) software.
For Cdc42-GTP pull-down experiment, a commercial kit (Cytoskeleton Inc.) was used following the manufacturer’s instructions, specifically using 107 B cells for 15 µg of beads. For immunoblotting, stimulated cells were then lysed in lysis buffer (20 mM Tris-HCL, pH 8.0,150 mM NaCl, 5 mM EDTA, Protease Inhibitor cocktail [Roche], 10 mM NaF, 1 mM Na3VO4, and 1% NP-40) for 15 min on ice, and samples were loaded into 4–20% (for Cdc42-GTP pull-down) or 12% (for all other applications) for electrophoresis. Proteins were detected with antibodies against pErk, pAkt (S473), pCD19, pSyk, pSrc, totalErk, pSTAT3 (Ser), and Cdc42 (all from Cell Signaling Technology) using the secondary HRP-conjugated anti–rabbit or anti–mouse antibodies (Jackson ImmunoResearch Laboratories).
Blot densitometry analysis was performed using the ImageJ (National Institutes of Health) software.
4
B Cell Signaling Pathway Analysis
5
Stimulation of Isolated B Cells
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Isolated B cells were maintained in a complete medium supplemented with 10 ng/ml of IL-4 and IL5 (Petrotech) and stimulated under different conditions: 1 or 5 μg/ml anti-IgM F(ab)2 fragment (Jackson ImmunoResearch), 0.3 μg/ml anti-CD40 (Thermo Fisher) or 3 μg/ml CpG (sigma).
6
B Cell Stimulation and Metabolic Modulation
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CFSE- or CTV–labeled cells at a concentration of 106 cells per mL were stimulated in complete B cell medium supplemented with combinations of 1 μg/mL LPS (Sigma) or 0.05 μg/ml CD40L (R&D Systems), 5 μg/ml anti-IgM F(ab)2 fragment (Jackson ImmunoResearch), 1.5 ug/mL CpG (Sigma) 10 ng/ml of IL-4 (R&D Systems), or 10 ng/ml of IL-5 (R&D Systems). CFSE or CTV dilution was measured after 3 or 4 days by flow cytometry. Various agents were added as follow: L-ascorbic acid (200 μM) on day1, Mitoqunione of specified concentrations, MitoTempo of specified concentrations, hemin (60 μM) on day 1 and Rapamycin (50 nM) on the specified time points.
7
Immunoblotting of B cell signaling
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Purified B cells were left at 37°C for 10 minutes in Imaging buffer (PBS, 0.5% FCS, 1 g/L D -Glucose, 2 mM MgCl2, and 0.5 mM CaCl2) to equilibrate before stimulation. They were then stimulated for various times with 5 μg/ml anti-IgM F(ab)2 fragment (Jackson ImmunoResearch) and 1.5 ug/mL CpG, 10 ng/ml of IL4, 10 ng/ml of IL-5, or coated microspheres (see previous section). For immunoblotting, stimulated cells were then lysed in lysis buffer (20 mM Tris-HCL, pH 8.0, 150 mM NaCl, 5 mM EDTA, Protease Inhibitor cocktail (Roche), 10 mM NaF, 1 mM Na3VO4, and 1% NP-40) for 30 minutes on ice, and samples were loaded into 12% PAGE gel (BIO-RAD) for electrophoresis. Proteins were detected with antibodies against p-Akt (Ser473), p-S6k1 (Thr389) and Erk using the secondary HRP-conjugated anti–rabbit or anti–mouse antibodies (see Key Resources Table). Blot densitometry analysis was performed using the ImageJ (National Institutes of Health) software.
8
B Cell Signaling Pathway Analysis
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Purified B cells were left at 37°C for 15 min in chamber buffer (PBS, 0.5% FCS, 1 g l−1 D-glucose, 2 mM MgCl2, 0.5 mM CaCl2) to equilibrate prior to stimulation. They were then stimulated for various times with 10 μg ml anti-IgM F(ab′)2 fragment (Jackson ImmunoResearch). Stimulated cells were lysed in lysis buffer [20 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA, Protease Inhibitor mixture (Roche), 10 mM NaF, 1 mM Na3VO4, 1% NP40] for 30 min on ice and samples loaded into Tris Glycin gels prior to electrophoresis using the miniprotean system (Bio-Rad). Proteins were detected with Abs against pErk, pAkt (S473), pCD19, pSyk, pSrc, total Erk (all from Cell Signaling Technology), tubulin (Sigma-Aldrich), or TC10 (Proteintech) using the secondary HRP-conjugated anti-rabbit or anti-mouse Abs (Jackson ImmunoResearch). Blot densitometry analysis was performed using Image J software.
9
Isolation and Activation of Mouse B Cells
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B cells from spleen or peripheral LNs were isolated using the B Cell Isolation Kit from Miltenyi Biotec (Cat. No. 130-090-862). B cells were cultured at a density of 0.5 × 106 cells per ml in RPMI-1640 medium (Dutch Modification) plus 10% foetal calf serum, antibiotics, 2 mM L-glutamine, 1 mM sodium pyruvate and β-mercaptoethanol (50 μM). Mitogen activation of B cells was achieved with LPS (10 μg/ml, Escherichia coli strain 0127:B8, Sigma Aldrich), anti-CD40 (10 μg/ml, clone 3/23; purified in-house) and/or anti-IgM (F(ab)2 fragment; 5 μg/ml, Jackson ImmunoResearch) in the presence of recombinant mouse IL-4 or hBaff (both from Peprotech).
In vitro derived GC B cells were generated as previously described30 (link). Briefly, 17.5 × 103 cells were co-cultured over a monolayer of 40LB stroma cells irradiated (120 Gy) and seeded in a 24-well plate 24 h before. IL-4 (2 ng/ml) was added to the RPMI medium that was replaced every 2 days. At day 4 of cell culture, iGC B cells were recovered and seeded on freshly irradiated 40LB stroma cells. At day 4, IL-4 (2 ng/ml) or IL-21 (10 ng/ml, Peprotech) was added to the media. Analysis of cell cycle was performed after adding 10 μM BrdU 30 min before harvest.
In vitro derived GC B cells were generated as previously described30 (link). Briefly, 17.5 × 103 cells were co-cultured over a monolayer of 40LB stroma cells irradiated (120 Gy) and seeded in a 24-well plate 24 h before. IL-4 (2 ng/ml) was added to the RPMI medium that was replaced every 2 days. At day 4 of cell culture, iGC B cells were recovered and seeded on freshly irradiated 40LB stroma cells. At day 4, IL-4 (2 ng/ml) or IL-21 (10 ng/ml, Peprotech) was added to the media. Analysis of cell cycle was performed after adding 10 μM BrdU 30 min before harvest.
10
Probing B-cell Signaling Pathways
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Purified B cells were left at 37°C for at least 10 min in the medium to equilibrate before stimulation. They were then stimulated for various times with 5 μg/ml anti-IgM F(ab)2 fragment (Jackson ImmunoResearch). For immunoblotting, stimulated cells were then lysed in lysis buffer (20 mM Tris–HCL, pH 8.0, 150 mM NaCl, 5 mM EDTA, protease inhibitor cocktail [Roche], 10 mM NaF, 1 mM Na3VO4, and 1% NP40) for 30 min on ice. The samples were loaded onto 12% PAGE gel (BioRad) for electrophoresis. Antibodies against p-CD19 (Tyr 531), p-Syk (Tyr 352), p-Akt (Ser 473), and p-Erk (Thr 202/Tyr 204) (Cell Signalling Technology), and secondary HRP-conjugated anti-rabbit antibody were used to probe B-cell signalling. Blot densitometry analysis was performed using ImageJ (National Institutes of Health) software.
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