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11 protocols using detachabead cd19

1

Isolation and Maintenance of Primary B Cells and EBV-Transformed Cell Lines

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Primary B cells were isolated from peripheral blood using CD19 Dynabeads and CD19 Detachabeads (Invitrogen), as previously described (27 (link)).
Cultures of mouse thymic stromal cells (OP9) expressing the human Delta-like ligand 1 (OP9-DLL1 cells) or control green fluorescent protein (GFP; OP9-GFP cells) (kindly provided by Graham Anderson, The University of Birmingham) were maintained for up to 20 passages in Opti-MEM medium (Gibco, Life Technologies) with 20% fetal calf serum (FCS).
BNHL-1 cells were derived from a bone marrow sample from a patient being treated for low-grade B cell non-Hodgkin's lymphoma (NHL). Cells of the BNHL-1 cell line were phenotypically the same as cells of the low-grade B cell NHL observed in the patient. These cells were infected with EBV and were continually maintained on the OP9-DLL1 cells, unless specified otherwise. An EBV-negative counterpart was maintained in the same manner.
293 cells containing recombinant wild-type EBV strain 2089 were maintained in RPMI, 10% FCS, and 100 μg/ml hygromycin. Akata cells containing recombinant wild-type EBV Akata were maintained in RPMI, 10% FCS, and 50 ng/ml G418.
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2

Isolation and Transformation of B-Cells

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Blood samples were collected from donors and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque centrifugation. Lymphoblastoid cell lines (LCLs) were generated by culturing PBMCs in standard LCL culture media (RPMI 1640 medium with 100 IU/ml penicillin, 100 mg/ml streptomycin, L-glutamine and 10% foetal calf serum) supplemented with 0.1ug/ml cyclosporin A (CSA; Sandimmune; Novartis Pharmaceuticals) with the addition of B95.8 strain EBV or a recombinant EBV lacking the BZLF1 gene (to generate B95.8 or BZLF1-K/O LCLs respectively). B-cells were isolated from PBMC immediately before use in experiments using anti-CD19 Dynabeads, and CD19 Detach-a-beads (Invitrogen) following the manufacturer’s instructions. B-cell purity was assessed by flow cytometry and purified B-cell preparations typically contained at least 95% B-cells.
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3

Isolation of Peripheral Blood B-cells

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Peripheral blood B-cells were isolated from healthy donors’ blood obtained from the Gulf Coast Blood Center (Houston, TX) by using CD19-positive magnetic beads and were released with the competitive CD19 DETACHaBEAD according to the manufacturer’s instructions (Invitrogen-Life Technologies, San Diego, CA). All procedures were performed under The University of Texas MD Anderson Cancer Center IRB-approved clinical protocol LAB08-0190.
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4

Cell Line Authentication and Lymphocyte Isolation

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Granta-519 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Remaining cell lines were obtained from the American Type Culture Collection (ATCC). Cells were maintained in RPMI-1640 (Hyclone) with 10% fetal bovine serum (Atlanta Biologicals) (RPMI-1640/10% FBS) in 5% CO2 atmosphere at 37°C. Cell lines were authenticated by STR analysis (MD Anderson Cancer Center Characterized Cell Line Core) and regularly tested for mycoplasma (Lonza).
Peripheral blood B-lymphocytes were isolated from healthy donors’ blood, obtained from Gulf Coast Blood Center (Houston, TX), with CD19-positive magnetic beads and released with the competitive CD19 DETACHaBEAD (Invitrogen).
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5

Mantle Cell Lymphoma Patient Samples

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Mantle cell lymphoma patients’ samples and clinical information were collected and published under The University of Texas MD Anderson Cancer Center IRB-approved clinical protocol LAB08-0190 for use of human tissues, with the written informed consent of all patients. Information about the patients is shown in Additional file 1: Table S1. Normal B lymphocytes were isolated from peripheral blood of healthy donors’ blood, obtained from Gulf Coast Blood Center (Houston, TX, USA), with CD19+ magnetic beads and released with DETACHaBEAD CD19 (Invitrogen, Grand Island, NY, USA).
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6

Isolation and Culture of Immune Cells

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PBMCs were isolated from anonymous healthy donor buffy coats provided by the Oslo University Hospital Blood Bank (project number: F8). NK cells were isolated from PBMCs using CD56-reactive microbeads and an AutoMACS Pro Separator according to the manufacturer’s instructions (Miltenyi Biotec, GmbH). Purification was verified by phenotypic analysis of the surface marker CD56. B and T cells were isolated from PBMCs using the Dynabeads™ CD19 PanB with DETACHaBEAD® CD19 and CD4-/CD8-Dynabeads™ positive isolation kits, respectively, as described by the manufacturer (Invitrogen Dynal AS, Oslo, Norway). The cells were cultured in either RPMI-1640 supplemented with 10% heat-inactivated FBS and 1% PS or in X-vivo 15 medium (Lonza, Basel, Switzerland) supplemented with 1% PS. Approval for obtaining buffy coats from healthy volunteers was granted by the Oslo University Hospital Ethics Committee.
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7

Isolation and Sorting of B Cell Subsets

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Mononuclear cells were isolated from PB and SF using a density centrifugation with LSM (MP Biomedicals, LLC, Santa Ana, CA, USA). B cells were isolated by positive selection with Dynabeads M450 CD19 and DETACHaBEAD CD19 (Invitrogen, Carlsbad, CA, USA) as previously described [19 (link)]. As we previously showed, only negligible levels of artificial activation of B cells occurred immediately after positive selection [20 (link)]. To avoid unwanted further stimulation, cells were normally rested on ice prior to any stimulation. Isolated B cells exhibited greater than 99.5 % viability and more than 95 % purity, confirmed by flow cytometry. Cells were stained with mouse or rabbit monoclonal antibody (mAb) against human CD19, IgD, CD27, CD80, CD86, CD183 (CXCR3), CD254 (RANKL) and CD307d (FcRL4) (all from BioLegend, San Diego, CA, USA). Naïve (IgD+CD27), IgD+-memory (IgD+CD27+), double-negative (IgDCD27) and switched-memory (IgDCD27+) B cell subsets were purified by flow cytometry.
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8

Isolation and Purification of CLL-B Cells

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The CLL-B cells were collected from blood samples at the Moores-UCSD Cancer Center in compliance with the Declaration of Helsinki and after approval of the UC San Diego Institutional Review Board (IRB) [24 ].Peripheral blood mononuclear cells (PBMC) from CLL patients were isolated using Ficoll-Hypaque gradient density centrifugation (Cat# 17-1440-03, GE Healthcare Life Science). For caspase activation assays, the CLL-B cells were purified by positive selection using Dynabeads CD19 pan B (Cat# 11143D, Invitrogen) and DETACHaBEAD CD19 (Cat# 12506D, Invitrogen) according to the manufacturer’s protocol. For the other assays, fresh or frozen PBMCs were used and cells were stained with CD19/CD5 antibodies for detection of double positive CLL-B cells.
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9

Isolation of B Cells and T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood using Ficoll–Paque gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA). CD19+ B cells were isolated by magnetic bead-based positive selection using Dynabeads and DETACHaBEAD CD19 (Invitrogen). CD4+ T cells were isolated with Dynabeads CD4 (invitrogen) and CD25+ T cells were depleted from the CD4+ T cell population using Dynabeads CD25 (invitrogen). All isolations were done according to the manufacturer’s instructions. The purity of isolated CD19+ B cells was consistently >95% as analyzed by flow cytometry.
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10

B Cell Isolation from Blood

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Using the same blood samples (both MCL patients and healthy controls) collected, B lymphocytes were isolated using CD19+ magnetic beads and the cells were released using DETACHaBEAD CD19 (Invitrogen). Samples with the purify and/or viability below 95% were selected for use.
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