Typically, freshly isolated or cryopreserved splenic B cells (1.6×105 per cm2) were cultured in BCM with IL-4 (10 ng/ml) and anti-CD40 (5 μg/ml, clone HM40-3, BioLegend) for 2 days prior to transduction. In some experiments, B cells were cultured with anti-CD40 and IL-4 for 3 or 4 days before transduction. Immature 2F5 KI B cells were cultured on 40LB cells plus IL-4 (2 ng/ml) for 2 days prior to transduction. Retrovirus was packaged in Gryphon Eco cells according to the manufacturer’s protocols. Virus-containing culture supernatant was harvested 48 hr post-transfection and was immediately mixed 1:1 with B cells in BCM containing 8 μg/ml Polybrene (MilliporeSigma), 10 ng/ml IL-21, and 3 μg/ml anti-CD40. Cells were spin-infected at 1258×g, 32°C, for 90 min, then incubated at 37°C. After 4.5 hr, 50% of the medium in each well was replaced with fresh BCM containing 10 ng/ml IL-21 and 3 μg/ml anti-CD40. The next day, B cells were washed in MACS buffer, and then plated on 40LB cells with 10 ng/ml IL-21.
Hm40 3
Manufactured by BioLegend
The HM40-3 is a laboratory equipment product manufactured by BioLegend. It is designed for use in scientific research and analysis. The core function of the HM40-3 is to perform specific tasks related to the handling and processing of biological samples. No further details on the intended use or capabilities of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Dynabead, by Thermo Fisher Scientific (2 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (2 mentions)
Polybrene, by Merck Group (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by BioLegend (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse il 2, by R&D Systems (1 mentions)
Rm134l, by BioLegend (1 mentions)
Ticagrelor, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Recombinant il 4, by BioLegend (1 mentions)
Anti cd19 mab, by Thermo Fisher Scientific (1 mentions)
48 well culture plate, by Corning (1 mentions)
Dead cell removal kit, by Miltenyi Biotec (1 mentions)
7 protocols using hm40 3
1
Retroviral Transduction of Splenic B Cells
2
Lymphocyte Populations Analysis by Flow Cytometry
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Lymphocyte populations were analysed by flow cytometry in single‐cell suspensions from spleen, PP, SI‐LP, peritoneal cavity and bone marrow using combinations of antibodies as previously described (Chapman et al, 2013 ).
For proliferation and class‐switch experiments, B cells were purified by negative selection from single‐cell suspensions from spleen using magnetic separation B‐cell isolation kit (Dynabeads; Invitrogen) according to the manufacturer's instructions. Purified cells (purity > 95%) were cultured for 4–5 days at 106 cells/ml in RPMI supplemented with 10% FCS and LPS (10 μg/ml; Sigma), IL‐4 (10 ng/ml; Biolegend), TGF‐β (2 ng/ml; eBioscience), IL‐5 (10 ng/ml; Biolegend) and/or anti‐mouse CD40 antibody (5 μg/ml, HM40‐3; Biolegend). For proliferation analyses, B cells were labelled with 5 μM CellTrace Violet (Molecular Probes) in PBS for 15 min at 37°C before culture. Flow cytometry analyses were performed in FACS buffer using the antibodies listed inAppendix Table S1 .
For proliferation and class‐switch experiments, B cells were purified by negative selection from single‐cell suspensions from spleen using magnetic separation B‐cell isolation kit (Dynabeads; Invitrogen) according to the manufacturer's instructions. Purified cells (purity > 95%) were cultured for 4–5 days at 106 cells/ml in RPMI supplemented with 10% FCS and LPS (10 μg/ml; Sigma), IL‐4 (10 ng/ml; Biolegend), TGF‐β (2 ng/ml; eBioscience), IL‐5 (10 ng/ml; Biolegend) and/or anti‐mouse CD40 antibody (5 μg/ml, HM40‐3; Biolegend). For proliferation analyses, B cells were labelled with 5 μM CellTrace Violet (Molecular Probes) in PBS for 15 min at 37°C before culture. Flow cytometry analyses were performed in FACS buffer using the antibodies listed in
3
Alloantigen-Specific Induction of iTregs
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For the alloantigen-specific system, 10×104 CD4+Foxp3− cells derived from Foxp3RFP x IL-10GFP double reporter mice (C57BL/6 background) or Foxp3hCD2 (BALB/c background) were plated with either mTECs (APC to T cell ratio 1:50 if not indicated otherwise) or DCs (APC to T cell ratio 1:10 if not indicated otherwise) in presence of 100 ng/ml recombinant mouse IL-2 (R&D systems) for six days. For in vitro blocking experiments, titrated amounts of anti-CD40 (HM40-3, Biolegend), anti-CD70 (FR70, Biolegend), anti-CD137 (TKS-1, Biolegend) and anti-OX40L (RM134L, Biolegend) was used. At the end of the cultures, allo-iTregs were either directly phenotypically analyzed or sorted as CD4+CD90+Foxp3+ cells for subsequent analyses on FACS Aria II using the Foxp3 reporter molecules RFP or hCD2.
4
Purification and Activation of Splenic B Cells
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Mouse splenic B cells were purified by CD43-depletion using magnetic-activated cell sorting (MACS; Miltenyi Biotec) according to the manufacturer’s protocol. To turn on LMP1 expression in vitro, B cells isolated from LMP1flSTOP mice were treated with TAT-Cre as described previously28 . For CD40 activation, purified B cells were cultured in the presence of 1 μg/ml anti-CD40 (HM40–3; Biolegend). Two days after TAT-Cre transduction or CD40 stimulation, cells were analyzed by FACS or used in other experiments. All B cells were cultured in DMEM medium supplemented with 10% FBS, 100 IU/ml penicillin, 10 mM HEPES, 1× non-essential amino acids, 1 mM sodium pyruvate and 50 μM β-mercaptoethanol.
5
Purification and Activation of Splenic B Cells
6
Selectin-mediated Immune Cell Interactions
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Tissue culture plates were coated with Fc-chimeric P-selectin (CD62P, BioLegend #755404), E-selectin (CD62E, BioLegend #755504), and L-selectin (CD62L, BioLegend #772806) for up to 3 hours under standard culture conditions before addition of cells for overnight culture at 20 μg/ml unless otherwise indicated. Tissue culture plates were coated with anti-mouse PSGL1 antibody (4RA10, BD Biosciences #557787) for up to 3 hours under standard culture conditions before addition of cells for overnight culture at 10 μg/ml, unless otherwise indicated. Blocking with anti-mouse CD40 (HM40-3, BioLegend #102908), CD40L (MR1, BioLegend #106508), MCP-1 (2H5, BioLegend #505905), CD41 (MWReg30, BioLegend #133910), or CD61 (2C9.G2, BioLegend #104310) and experiments with Ticagrelor (Sigma-Aldrich) and DKK-1 (BioLegend #759604) were performed by addition to PBMCs before overnight culture. Thrombin (Sigma-Aldrich) was added to the collected PBMCs for 30 min before analysis.
7
In Vitro B Cell Differentiation from Lung Tissue
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Dead cells among the lymphocytes isolated from lung tissues were removed by using the Dead Cell Removal Kit (Miltenyi Biotec) for in vitro differentiation into CD23+ B cells. CD19+ B cells were obtained through positive selection by using the Dynabeads (Invitrogen) and an anti-CD19 mAb (eBioscience). CD19+ B cells were dispensed in triplicates (3.5 × 105 cells/well) from 8-week-old three control mice or pSS model mice using 48-well culture plate (Corning Incorporated). B cells were cultured for 1 week in Roswell Park Memorial Institute (RPMI) 1640 containing 10% FBS with an anti-CD40 mAb (5 µg/ml; BioLegend, HM40-3) and recombinant IL-4 (100 ng/ml; BioLegend) as described in a previous report (27 (link)).
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