Dynabeads cd19 pan b

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.

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.

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.

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).

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.