B cells isolated from human PBMCs by Magnetic-Activated Cell Sorting (17954, StemCell Technologies) were immobilized onto glass slides coated with poly-L-lysine (Sigma-Aldrich). The BCR was labeled with a human IgM+G antibody (Alexa Fluro 546-(Fab)2-anti-Ig(M+G)). This antibody was generated from the F(ab′)2 fragment (Jackson ImmunoResearch, West Grove, PA) using a published protocol (23 (link)). Alexa Fluro 546-(Fab)2-anti-Ig(M+G) was incubated with B cells for 30 min on ice before Fc receptor blocking (422301; BioLegend). Then, the B cells were stimulated for 0, 5, 15, or 30 min at 37°C before fixation with 4% paraformaldehyde. In some experiments, B cells were stimulated for 48 h at 37°C with CpG (10 μg/mL) and CD40L (1 μg/mL) to detect activation of phospho-STAT3. The following specific antibodies were used to stain the cells after permeabilization with 0.05% saponin (Sigma): anti-CD38 (ab235118; abcam), anti-CD24 (ab202073; abcam), anti-phospho-STAT3 (9145S; Cell Signaling Technology), anti-phosphotyrosine (05-1050X; Merck Millipore), anti-pBTK (ab52192; abcam), and anti-pCD19 (3571S; Cell Signaling Technology). Images were obtained under a confocal microscope (Nikon A1R) using 405, 488, 546, 647 nm lasers. Colocalization and MFI were determined by the NIS-Elements AR 3.2 software.
F ab 2 fragment
Manufactured by Jackson ImmunoResearch
Sourced in United Kingdom
F(ab')2 fragment is a laboratory product that is derived from an antibody. It consists of the antigen-binding regions (Fab) of the antibody connected by a hinge region, but lacks the Fc region. The F(ab')2 fragment retains the ability to bind to antigens, but does not have the effector functions associated with the Fc region.
Automatically generated - may contain errors
Lab products found in correlation
Maleimide activated biotin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (2 mentions)
Idelalisib, by Selleck Chemicals (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab202073, by Abcam (1 mentions)
Ab52192, by Abcam (1 mentions)
Magnetic activated cell sorting (macs), by STEMCELL (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
Biotin quantification kit, by Thermo Fisher Scientific (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Molecular probes protein labeling kits, by Thermo Fisher Scientific (1 mentions)
Smifh2, by Merck Group (1 mentions)
Alexa fluor 594 anti mouse cd19 antibody, by BioLegend (1 mentions)
Anti p53, by Santa Cruz Biotechnology (1 mentions)
Anti mdm2, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
F ab 2 fragments, by Southern Biotech (1 mentions)
Facsaria sorp, by BD (1 mentions)
10 protocols using f ab 2 fragment
1
Visualizing B Cell Receptor Activation
2
Generation of Mesothelin-Specific T Cells
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To generate mesothelin-specific T cells, αCD3/ICOS-activated, sorted CD4+ and bulk CD8+ T cells were transduced with a lentiviral vector encoding a chimeric anti-mesothelin single-chain variable fragment fusion protein containing the TCRζ signaling domain (first-generation meso-CAR) or a truncated CD3ζ nonsignaling domain (Δζ) that was generated as described previously (19 (link)). CAR expression was determined using a flow cytometry antibody specific for the murine F(ab')2 fragment (Jackson ImmunoResearch, 115-606-006). Cells were normalized for CAR expression before adoptive transfer.
3
Activating B cells using Fab' fragments and lipid bilayers
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The planar lipid bilayer and liposomes were prepared as described previously (20 (link)). The F(ab’)2 fragment (Jackson ImmunoResearch Laboratories) was used for making the monobiotinylated Fab’ fragment of anti-mouse IgM + G Ab (mB-Fab’-anti-Ig) following a published protocol (40 (link)). To break the disulfide bond that links the two Fab’, 20 mM 2-mercaptoethylamine was used, and the reduced cysteine was biotinylated by maleimide-activated biotin (Thermo Fisher Scientific). Next, Fab’ was purified, quantified, and labeled with Alexa Fluor 546 (Thermo Fisher Scientific). To activate B cells with sAg, splenic B cells were labeled with AF546-mB-Fab’-anti-Ig (2 μg/ml) and added in mB-Fab’-anti-Ig (8 μg/ml) for 30 min and streptavidin (1 μg/ml) for 10 min at 4°C. As a control, streptavidin was omitted. Cells were incubated at 37°C for indicated times. To activate B cells with mAg, cells were incubated with AF546-mB-Fab’-anti-Ig and mB-Fab’-anti-Ig tethered to planar lipid bilayers by streptavidin at 37°C for varying lengths of time.
4
Mono-biotinylated Anti-mouse Fab' Fragment Generation
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Mono-biotinylated Fab’ fragment of anti-mouse antibody (Ab) (mB-Fab’-anti-IgG+M) was generated from the F(ab’)2 fragment (Jackson ImmunoResearch) using a published protocol (45 ). The disulfide bond that links the two Fab’ fragments was reduced using 2-mercaptoethylamine, and the reduced cysteine was biotinylated by maleimide-activated biotin (Thermo Scientific). Fab’ was further purified using Amicon Ultra centrifugal filters (Millipore). One biotin per Fab’ was confirmed using a biotin quantification kit (Thermo Scientific). Fab’ was labeled with Alexa Fluor 546 (AF546-mB-Fab’-anti-IgG+M) based on the manufacturer-recommended protocol (Invitrogen).
5
Inhibiting Cytoskeletal Regulators in Cells
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For inhibition of formin and Arp2/3, cells were incubated with inhibitors for 5 min at 37 °C before being added to the imaging chamber, which had the inhibitor at the same concentration used for incubation. SMIFH2 (Sigma-Aldrich) was used at a 25 μM concentration. Arp2/3 complex inhibitor I, CK666 (Calbiochem) was used at 50 μM. For N-WASP inhibition, wiskostatin B (EMD Bioscience) was used at 10 μM to incubate cells for 1 h at 37 °C. Mono-biotinylated fragment of antibody (mbFab′-anti-Ig) was generated from the F(ab′)2 fragment (Jackson Immuno Research, West Grove PA) using a published protocol51 (link). Fcγ RIIB (CD32) antibody (Cat# 553141, BD Biosciences) was conjugated with Alexa Fluor 546 using Molecular Probes Protein labeling kits (Cat# A10237, Invitrogen) following manufacturer protocols. For labeling of CD19 we used the Alexa Fluor 594 anti-mouse CD19 antibody at 0.15 μg/ml (Cat# 115552, BioLegend).
6
Comprehensive Western Blot Assay Protocol
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Western blotting was performed exactly as described27 (link). The primary antibodies used were: anti-β-actin (Sigma, Deisenhofen, Germany; A5316), anti-MDM2 (Santa Cruz, Heidelberg, Germany (sc-965)), anti-p21Cip/Waf (Santa Cruz; sc-397), anti-p53 (Santa Cruz; sc-126), and anti-GAPDH (Sigma; G9545). Secondary antibody F(ab′)2 fragments coupled to horseradish peroxidase and specific for rabbit-IgG (No. 111-036-045) or mouse-IgG (No. 115-036-072) were obtained from Jackson ImmunoResearch, Newmarket, UK. A freshly made solution of luminol (2.5 mM), p-coumaric acid (0.2 mM) and H2O2 (0.01%) in 100 mM Tris-HCl (pH 8.8) was used as reagent for chemiluminescent detection.
7
In Vitro B Cell Activation Assay
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For mouse in vitro activation studies, FO B cells and MZ B cells were sorted using the FACSAria or FACSAria SORP (BD). For dead cell exclusion, 20 ng/mL DAPI (Thermo Fisher Scientific) was added to the cells just prior to sorting. FO B cells and MZ B cells were cultured in RPMI medium plus penicillin-streptomycin, l -glutamine, and 10% FBS and stimulated with polyclonal goat anti–mouse IgM (Southern Biotech; 0.6–150 μg/mL), or with equimolar concentrations of the Fab′2 fragments (Southern Biotech; 0.4–100 μg/mL) for 20 hours, after which activation was measured using flow cytometry as described above.
For human in vitro activation studies, naive and MZ-like B cells were sorted using the BD FACSAria system. Cells were cultured in RPMI medium plus penicillin-streptomycin,l -glutamine, and 10% FBS and stimulated with polyclonal goat anti–human IgM (Jackson ImmunoResearch, AffiniPure; 0.6–75 μg/mL), or with equimolar concentrations of the Fab′2 fragments (Jackson Immunoresearch Affinipure; 0.4–50 μg/mL) for 20 hours, after which activation was measured using flow cytometry as described above.
For human in vitro activation studies, naive and MZ-like B cells were sorted using the BD FACSAria system. Cells were cultured in RPMI medium plus penicillin-streptomycin,
8
Measuring hERG Endocytic Recycling
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hERG endocytic recycling was measured using a modified sandwich cell-surface ELISA assay described previously37 (link). Briefly, cell-surface hERG was labelled with anti-HA primary antibody (1 h, on ice). Ab-hERG complexes were then internalized for 20 minutes at 37 °C; complexes remaining on the cell surface were then blocked with mouse monovalent F(ab′)2 fragments (1:100; Jackson ImmunoResearch Laboratories, 1 h on ice). Recycling of the internalized Ab-hERG complexes was enabled by incubating cells at 37 °C for 0–20 minutes. Exocytosed Ab-hERG complexes were detected with HRP-conjugated secondary F’(ab)2 as described above. Background signal was measured using a non-specific primary Ab as described above. Blocking efficiency with mouse monovalent F(ab′)2 fragment was determined to be over 95% (data not shown). The size of the endocytic hERG pool following 20-minute incubation at 37 °C was measured in parallel, with recycling efficiency being expressed as percent of this pool.
9
Evaluating PI3K Isoform Inhibitors in MCL
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The expression of PI3Kγ (p110γ) on the MCL cell lines and PBMC from MCL patients was analysed by western blotting. Primary cells were purified by MACS (Miltinyi Biotech, Woking) to remove non-B cells as T cells and monocytes are known to express PI3Kγ (> 99% purity; data not shown). The monocyte cell line THP-1 was used as a positive control for PI3Kγ expression.
In order to determine the efficacy of the PI3K inhibitors MCL cells were incubated for 1 h with inhibitors to different PI3K isoforms [PI3Kα (A66), PI3Kδ (idelalisib), PI3Kγ (CZC24832) (all from Selleck, Cambridge UK), and the dual PI3Kδ/γ inhibitor [(duvelisib) Verastem Oncology]. The effects of the inhibitors on the phosphorylation of AKT in response to BCR cross-linking with goat anti-human IgM [F(ab)2 fragments; 20 μg/ml; Jackson Immunoresearch, Pensylvania)] and chemokine signalling with CLL21 [(1 µM) Biotechne, Abbingdon] were then examined by western blotting. Inhibitors were titrated using the following concentrations 100 nM; 500 nM; 1 μM and 2 μM. AKT phosphorylation was inhibited by 1 μM (Supplementary Figure1 F) which is below the peak plasma concentration for idelalisib and duvelisib33 (link),34 (link).
In order to determine the efficacy of the PI3K inhibitors MCL cells were incubated for 1 h with inhibitors to different PI3K isoforms [PI3Kα (A66), PI3Kδ (idelalisib), PI3Kγ (CZC24832) (all from Selleck, Cambridge UK), and the dual PI3Kδ/γ inhibitor [(duvelisib) Verastem Oncology]. The effects of the inhibitors on the phosphorylation of AKT in response to BCR cross-linking with goat anti-human IgM [F(ab)2 fragments; 20 μg/ml; Jackson Immunoresearch, Pensylvania)] and chemokine signalling with CLL21 [(1 µM) Biotechne, Abbingdon] were then examined by western blotting. Inhibitors were titrated using the following concentrations 100 nM; 500 nM; 1 μM and 2 μM. AKT phosphorylation was inhibited by 1 μM (Supplementary Figure
10
Primary B Cell Isolation and Culture
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Primary B lymphocytes (normal and CLL) were used in all the experiments described. Cells were isolated from peripheral blood and buffy coats by centrifugation over Lymphoprep (Axis-Shield, Oslo, Norway). Normal B cells were purified by negative selection using a B cell isolation kit (Miltenyi Biotec, Bisley, U.K.) (>98% CD19+). All cultures involving primary lymphocytes employed RPMI 1640 medium containing 1% fatty acid-free BSA (Sigma-Aldrich, Poole, U.K.); HUVEC were cultured in IMDM containing 20% FCS, whereas HS-5 and CD40L-transfected fibroblasts were cultured in DMEM containing 10% FCS. All culture media were supplemented with 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Paisley, Scotland). Ibrutinib, fostamatinib, and idelalisib were all used at 1 μM (Selleckchem). These drug concentrations were sufficient to maximally inhibit the respective target kinases following BCR ligation (data not shown); IC50 values and peak plasma concentrations are shown in Supplemental Table II . Goat anti-human IgM [F(ab)2 fragments (Jackson ImmunoResearch Laboratories)] was used at 20 μg/ml.
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