Eμ-TCL1 mice on C57BL/6 background were kindly provided by Carlo Croce (Ohio State University). C57BL/6 wild-type (WT) mice were purchased from Charles River Laboratories (Sulzfeld, Germany). Adoptive transfer of Eµ-TCL1 tumors was performed as previously described [42 (link),43 (link)]. Briefly, 1–2 × 107 B-cells enriched from Eμ-TCL1 splenocytes were transplanted intraperitoneally (i.p.) into C57BL/6N WT animals. B-cell enrichment was performed using EasySep™ Mouse Pan-B Cell Isolation Kit (Stemcell Technologies, Vancouver, BC, Canada), yielding a purity above 95% of CD5+ CD19+ cells. Tumor load was assessed in the blood every week using flow cytometry as the proportion of CD5+ CD19+ cells among CD45+ cells. Animals with a tumor load >90% in peripheral blood were sacrificed; spleen was isolated and mechanically dissociated in PBS. All animal experiments were carried out according to institutional and governmental guidelines approved by the local authorities (Regierungspräsidium Karlsruhe, permit number G98/16, approved on 13 July 2016).
Easysep mouse pan b cell isolation kit
Manufactured by STEMCELL
Sourced in Canada
The EasySep Mouse Pan-B cell Isolation Kit is a lab equipment product designed for the isolation of mouse B cells from a single-cell suspension. The kit uses an antibody-based selection method to target and separate the B cells from the sample.
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Lab products found in correlation
Shandon cytospin 4, by Thermo Fisher Scientific (3 mentions)
Anti cd19 pe, by BD (1 mentions)
Sector imager 2400 reader, by Mesoscale (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Nicotine, by Merck Group (1 mentions)
Duolink in situ detection reagent, by Merck Group (1 mentions)
3h thymidine, by PerkinElmer (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Block it alexa fluor red fluorescent control, by Thermo Fisher Scientific (1 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Countess 2 fl, by Thermo Fisher Scientific (1 mentions)
Pan b cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Tak 243, by MedChemExpress (1 mentions)
Mg132, by Merck Group (1 mentions)
Antimouse cd23 pe, by BD (1 mentions)
Anti mouse cd19 pe cy7, by BD (1 mentions)
Easysep release mouse pe positive selection kit, by STEMCELL (1 mentions)
10 protocols using easysep mouse pan b cell isolation kit
1
Adoptive Transfer of Eμ-TCL1 Tumors
2
Isolation and Analysis of Primary B-Cells
3
Measuring TNF mRNA and NF-κB Translocation in B Cells
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For measurements of TNF mRNA, FO B cells were sorted (the gating strategy is shown in fig. S4A) and stimulated with 2.5 μM CpG (InvivoGen) and nicotine (Sigma-Aldrich) in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin and 10 mM Hepes. After 4 hours of incubation, cells were collected, and pellets were solubilized in TRIzol reagent. These solution were stored at −80°C. For measurement of secreted TNFα, the culture supernatant was collected after 24 hours of incubation. TNFα concentration was measured with the V-PLEX Mouse TNF-α Kit (Meso Scale Diagnostics) and the Sector Imager 2400 Reader (Meso Scale Diagnostics) according to the manufacturer’s instructions.
For NF-κB translocation assays, B cells were isolated from naïve mouse spleens with the EasySep Mouse Pan-B cell Isolation Kit (STEMCELL Technologies) and suspended at 1.0 × 106 cells/ml in the supplemented RPMI 1640 (Gibco). Cells were stimulated with 1.0 μM CpG with or without nicotine for an hour. After washing, cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer.
For NF-κB translocation assays, B cells were isolated from naïve mouse spleens with the EasySep Mouse Pan-B cell Isolation Kit (STEMCELL Technologies) and suspended at 1.0 × 106 cells/ml in the supplemented RPMI 1640 (Gibco). Cells were stimulated with 1.0 μM CpG with or without nicotine for an hour. After washing, cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer.
4
Proximity Ligation Assay for B Cell Signaling
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B cells were isolated from naïve mouse spleens with the EasySep Mouse Pan-B cell Isolation Kit (STEMCELL Technologies) and stimulated following the same method as BCR signal analyses described above. The proximity of the interested molecules was visualized using Duolink In Situ Detection reagents (Millipore Sigma) according to the manufacturer’s instructions. Briefly, fixed cells were blocked for 50 min at 37°C and labeled with antibodies listed in table S1 for 30 min at room temperature in 96-well V-bottom plates. After a washing step, PLA probes were added in wells and incubated for an hour at 37°C. Cells were subsequently washed and incubated with ligase for 30 min at 37°C, followed by oligonucleotide amplification with DNA polymerase for 100 min at 37°C. Cell suspensions were applied to slide glasses using SHANDON Cytospin 4 (Thermo Fisher Scientific) and observed with a Zeiss Apoptome 3 microscope (Zeiss).
5
B Cell Polyclonal Activation Assay
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To assess the polyclonal activation of B cells in WT and Shp1-Tg genotypes, we set up B cell cultures from WT, Shp1-Tg+/+, and Shp1-Tg+/− mice and used anti-mouse IgM for activation (anti-mouse IgM F (ab’)2, μ chain-specific, Functional Grade Purified, eBioscience, cat# 16-5092). Spleen cells of n = 3–6 mice per genotype were harvested. B cells were isolated using immunomagnetic negative selection (EasySep mouse pan B cell isolation kit, StemCell Technologies).
For proliferation assays, B cells were suspended in DMEM supplemented with 10% FBS and seeded in 96-well culture microplates at a density of 2 × 105 viable cells in 200 μl of medium per well. Anti-IgM (10 μg/ml) was added to stimulated wells, while control wells contained only medium and the cells (both in triplicates). The culture was maintained for 5 days. [3H] thymidine (Perkin Elmer) incorporation was measured as described for T cells. Stimulation index (SI) was calculated by dividing the mean cpm values of stimulated wells with the mean cpm values of control wells.
For proliferation assays, B cells were suspended in DMEM supplemented with 10% FBS and seeded in 96-well culture microplates at a density of 2 × 105 viable cells in 200 μl of medium per well. Anti-IgM (10 μg/ml) was added to stimulated wells, while control wells contained only medium and the cells (both in triplicates). The culture was maintained for 5 days. [3H] thymidine (Perkin Elmer) incorporation was measured as described for T cells. Stimulation index (SI) was calculated by dividing the mean cpm values of stimulated wells with the mean cpm values of control wells.
6
B Cell Isolation and siRNA Transfection
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B cells were isolated from naïve mouse spleens with the EasySep Mouse Pan-B cell Isolation Kit (STEMCELL Technologies) and suspended in Opti-MEM I Reduced Serum Medium (Gibco). Each nAChR siRNA or control siRNA was cotransfected with BLOCK-iT Alexa Fluor Red Fluorescent Control (Thermo Fisher Scientific) to the cells using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific), according to the manufacturer’s instruction. Alexa Fluor 555–positive cells were sorted out by flow cytometry after 4 hours of incubation. Cells underwent further assays after additional 20 hours of culture in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 10 mM Hepes. siRNAs used are listed in table S1.
7
Isolation of Murine B Cells
8
In Vitro Stimulation of Splenic B Cells
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Splenic B cells were negatively enriched using the EasySep Mouse Pan-B cell Isolation Kit (StemCell Technologies). For the in vitro stimulation assay, 3 × 106 B cells were treated for 6 h at 37°C with tunicamycin (33.3 μg/ml) (#12819S; CST), MG132 (10 μM) (#M7449; Sigma-Aldrich), and TAK-243 (500 nM) (# HY-100487; MedChemExpress). Then, the cells were washed twice in sterile PBS to remove residual proteins in the culture media. Proteins levels were measured using immunoblot.
9
Purification of Murine B1 and B2 Cells
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Cells were isolated from the spleen, bone marrow, or peritoneal cavity as described.29 , 30 B1 cells and B2 cells were purified from the peritoneal cavity cells. In brief, total B cells were first purified using Easysep mouse pan‐B cell isolation kit (STEMCELL Technologies, Vancouver, BC, Canada). Next, B1 cells (CD23−) and B2 cells (CD23+) were enriched from purified B cells using Easysep release mouse PE positive selection kit (STEMCELL) in combination with antimouse CD23‐PE (BD Biosciences). The efficiency of cell enrichment was evaluated by cytometry using antimouse CD19‐PE‐Cy7 (BD Biosciences) and antimouse CD23‐PE. Purity of B1 cell and B2 cell suspension was respectively about 85%‐91% and >98%.
10
Adoptive Transfer of Leukemic Cells
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Adoptive transfer was performed by i.p. transplantation of 1–2 × 107 leukemic Eµ-TCL1 splenocytes into respective, syngeneic animals to avoid tumor rejection [14 (link)]. Splenocytes were enriched for CD19+ cells using the EasySep™ Mouse Pan-B Cell Isolation Kit (Stemcell Technologies, Cologne, Germany), yielding a purity of above 95% CD5+ CD19+ cells.
All animal experiments were carried out according to institutional and governmental guidelines approved by the local authorities (Regierungspräsidium Karlsruhe, permit numbers: G123/14, G36/14, G98/16, G53/15). Eµ-TCL1 (C. Croce, OH, USA) mice were held in specific pathogen-free conditions on a pure C57BL/6 N or J background at the central animal facility of the German Cancer Research Center (DKFZ).
All animal experiments were carried out according to institutional and governmental guidelines approved by the local authorities (Regierungspräsidium Karlsruhe, permit numbers: G123/14, G36/14, G98/16, G53/15). Eµ-TCL1 (C. Croce, OH, USA) mice were held in specific pathogen-free conditions on a pure C57BL/6 N or J background at the central animal facility of the German Cancer Research Center (DKFZ).
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