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Dynabeads cd19 pan b

Manufactured by Thermo Fisher Scientific
Sourced in United States

Dynabeads CD19 Pan B is a magnetic bead-based product for the isolation of human B cells from a variety of biological samples. The beads are coated with antibodies specific to the CD19 antigen, which is expressed on the surface of B cells. This product can be used to positively select or enrich B cells for further analysis or downstream applications.

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12 protocols using dynabeads cd19 pan b

1

Isolation of T Cells from PBMCs

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Peripheral blood mononuclear cells (PBMCs) from healthy donors were separated by Ficoll-Paque (GE Health Care #17-1440-02) density gradient separation. T cells from PBMCs were enriched, first by depleting B cells (Dynabeads CD19 Pan B (Invitrogen #11143D)), followed by monocyte depletion by plastic adhesion.
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2

Isolation and Culture of EBV-associated Cells

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Cell lines include EBV-positive Raji cells (ATCC CCL-86) [59 (link)], EBV-negative Elijah B cells (kindly provided by Prof. A.B. Rickinson), 293 cells (ATCC CRL-1573) [60 (link)], T cells specific for EBNA1 3E10, EBNA3C 5H11, gp350 1D6 and BNRF1 VSD epitopes (kindly provided by Prof. J. Mautner) and autologous LCLs (kindly provided by Prof. J. Mautner). Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus and primary B cells were isolated using Dynabeads CD19 Pan B (Invitrogen) and DETACHaBEAD CD19 kit (Invitrogen). RPMI containing 10% fetal calf serum (F7524, Sigma) was used to culture 293, Raji and Elijah cells. T-cell clones and lines were cultured as previously described [17 (link),35 (link)].
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3

Leukocyte Fractionation and DNA Extraction from Breast Cancer

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Peripheral blood specimens were collected from 10 newly diagnosed female BC patients from the Tumor Hospital of Harbin Medical University and from 10 healthy female volunteers from Harbin Medical University. Leukocytes were freshly isolated from peripheral blood using a Human Peripheral Blood Leukocyte Isolation Kit (Solarbio, Beijing, China) within two hours after blood collection. Isolated leukocytes were suspended in 1 mL PBS for the following cell fractionations. B cells were isolated from leukocytes first using Dynabeads® CD19 pan B (Invitrogen, Carlsbad, CA, USA). Then, monocytes were isolated from B cell-depleted leukocytes using Dynabeads® CD14 (Invitrogen, Carlsbad, CA, USA). Finally, T cells were purified from B-cell/monocyte-depleted leukocytes using Dynabeads® CD3. DNA extraction and bisulfite conversion from B cells, T cells, and monocytes were performed immediately after purification and the experimental procedures have been described above.
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4

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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5

CD19+ B Cell Isolation and EBV Transformation

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CD19+ B cells were isolated from PBMCs using Dynabeads CD19+ pan B (11143D, Invitrogen) according to the manufacturer’s instructions. 2.5 × 108 cells of PBMCs were resuspended in 10 ml isolation buffer (PBS, 0.1% BSA, 2 mM EDTA). 250 μl of prewashed beads were added to PBMCs and incubated for 20 min in 4°C with gentle rotation. For positive isolation of CD19+ B cells, beads and supernatant were separated using magnet, and supernatant was discarded. Beads were washed three times, and beads bounded with CD19+ B cells were resuspended with 2.5 ml of cell culture medium (80% RPMI 1640, 20% heat-inactivated FBS, glutamine). CD19+ B cells were released from Dynabeads using DETACHaBEAD (Invitrogen, ca12506D) according to the manufacturer’s instruction.
B cells were infected with EBV to transform lymphocytes. 10 ml of B cells were transferred into a T75 flask. 1.5 ml of stock EBV collected from a B95-8 strain-containing marmoset cell line and 1 ml of phytohemagglutinin P (PHA-P) were added to flask and incubated in 37°C with a 5% CO2 atmosphere incubator. Every 5–7 d, 10 ml of cell culture medium was added. Cells were let to grow in the CO2 atmosphere incubator for 30 d until all B cells were transformed to LCLs.
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6

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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7

Isolation and Purification of Tumor Cells

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Tumour cells were isolated using Dynabeads® CD19 Pan B (number 11143D, Thermo Fisher Scientific, Waltham MA, USA) combined with DETACHaBEAD® CD19 (number 12506D, Thermo Fisher Scientific, Waltham MA, USA). Subsequently, the cell suspensions were depleted of naïve B-cells using anti-IgD coated Dynabeads (the tumour cells were IgD negative as assessed before by immunohistochemistry on frozen tissue sections). The purity of the tumour cell fraction was checked by flow cytometry for expression of CD20, κ and λ (IQ Products, Groningen, The Netherlands). After purification, cells were washed three times with cold PBS and centrifuged at 1200 rpm for 5 minutes at 4°C, resuspended in lysis buffer (Cell signalling technologies, Danvers, USA, #9803) and placed on ice for 30–45 min. The supernatant containing mostly membrane and cytoplasmic proteins (nuclei are not efficiently lysed in this buffer) was collected by centrifugation at 14.000 rpm for 10 minutes at 4°C and 20-fold concentrated with the Vivaspin® 2 Centrifugal Concentrator. The protein concentration was measured using the Pierce BCA Protein Assay Kit (#23227; Thermo Scientific, Waltham MA, USA).
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8

Isolation and Purification of B-cells from PBMCs

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PBMCs were isolated by Ficoll density centrifugation and were stored in liquid nitrogen. Cryopreserved PBMCs were thawed in a 37 °C water bath and when the sample started to melt, then warm RPMI medium with supplements (10% AB serum, 2 mM l-glutamine, 100 IU/ml penicillin, 100 IU/ml streptomycin) was added. B-cells were isolated trough positive selection using superparamagnetic polystyrene beads (Dynabeads™ CD19 Pan B, number 11143D, Thermo Fisher Scientific) coated with a monoclonal antibody specific for the CD19 antigen and beads were then removed using DETACHaBEAD® CD19 kit (number 12506D, Thermo Fisher Scientific) according to the manufacturer's instructions. The purity of B-cells was confirmed by the flow cytometry where B-cells were determined by the expression of CD20 and the lack of CD3 and the purity was at least 97%. Purified B-cells were resuspended in RLT Plus buffer (Qiagen) to lyse the cells and protect the RNA from degradation.
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9

Activation and Analysis of NHL T Cells

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T cells from NHL tumors were enriched by depletion of CD19+ B cells using Dynabeads CD19 Pan B (Thermo Fisher) according to the manufacturer’s protocol. Cytokine production was then activated for 6 hours using Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher) in a 1:1 bead-to-cell ratio, with GolgiPlug (BD Biosciences) present for the last 4 hours. Cells were fixed in 1.6% PFA to stop activity, followed by centrifugation and permeabilization in >90% ice-cold methanol. Samples were stored at −80°C before staining with antibodies and flow cytometry acquisition.
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10

T Cell Activation and Conditioned Medium Preparation

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PBMCs were separated from buffy coats of healthy blood donors by Ficoll-Paque density gradient centrifugation. T cells from PBMCs were enriched by negative selection using CD19 Pan B Dynabeads (ThermoFisher Scientific; Cat. No.: 11143D) according to the manufacturer`s instructions, followed by the removal of cells adherent to tissue culture dishes. T cells were seeded at 106 cells/ml density in complete or StemSpan SFEM medium and were left untreated or activated for 48 hours by Human T-Activator CD3/CD28 Dynabeads (ThermoFisher Scientific; Cat. No.: 11161D; 1 bead/1 T cell). Conditioned medium was purified by pelleting the cells with centrifugation, followed by magnetic cleanup of beads and filtration of the supernatants through a 0.22 µm pore size filter.
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