Blood from healthy donors was obtained from apheresis leukoreduction collars (Brigham and Women's Hospital, Crimson Core). Peripheral blood mononuclear cells were extracted using centrifugation (35 minutes, 400 × g) through Ficoll Paque (Sigma-Aldrich, GE17–1440–02). T cells were isolated using the EasySep Human T-cell Isolation Kit (StemCell Technologies, 17951) and the Big Easy magnet (StemCell, 18001), according to the manufacturer's protocol. T cells were stimulated with CD3/CD28-coated Dynabeads (Thermo Fisher Scientific, 11131D) and maintained in R10 supplemented with 20 U/mL IL2. 24–30 hours after isolation, T cells were infected with MSLN CAR by adding lentivirus dropwise to each well at an MOI of 5–6; infection efficiencies ranged from approximately 50% to 75%. 7 days after isolation, Dynabeads were removed; 14 days after isolation, T cells were frozen in 90% FBS supplemented with 10% DMSO. Fifteen to 20 hours before plating a coculture, T cells were thawed and cultured in R10 sans IL2.
Cd3 cd28 coated dynabeads
Manufactured by Thermo Fisher Scientific
Sourced in Austria
CD3/CD28-coated Dynabeads are magnetic beads coated with antibodies against the CD3 and CD28 receptors on T cells. These beads are designed to activate and expand T cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque, by Merck Group (1 mentions)
Big easy magnet, by STEMCELL (1 mentions)
Easysep human t cell isolation kit, by STEMCELL (1 mentions)
Imdm medium, by Thermo Fisher Scientific (1 mentions)
Human serum, by GE Healthcare (1 mentions)
Octomacs separator, by Miltenyi Biotec (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Quadromacs separator, by Miltenyi Biotec (1 mentions)
3 protocols using cd3 cd28 coated dynabeads
1
Isolation and Transduction of Primary T Cells
2
T cell expansion from peripheral blood
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Pure T cells were obtained from each patient by in vitro expansion of T cells from PB (or BM). Monocytes were first depleted by adherence to tissue culture flasks. The remaining cells were cultured for 14 to 21 days in IMDM medium (Life Technologies) supplemented with 10% human serum (PAA Laboratories GmbH, Pasching, Austria), interleukin-2 (100 IU ml−1) and CD3/CD28-coated Dynabeads (Thermo Fisher). The purity of the T cells was measured by flow cytometric analysis using the CD3 surface marker. When the purity of the T cells exceeded 95%, DNA was isolated using the NucleoSpin Blood QuickPure kit.
3
Isolation and Expansion of Murine γδ T Cells
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Single cell suspensions were generated from pooled lymph nodes (axillary, brachial, inguinal) and spleens of two WT mice as above. γδ T cells were isolated from this suspension using the γδTCR + T cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. In brief, CD3 + T cells were enriched by negative selection on a LD column and QuadroMACS separator (Miltenyi Biotec), before positive selection of γδ T cells using MS columns with an OctoMacs separator (Miltenyi Biotec). To yield a purer γδ T cell population, positive selection was performed twice. γδ T cells were cultured in IMDM medium supplemented with 10% FCS, 100U/mL penicillin/streptomycin, 2mM glutamine, 50μM 2-Mercaptoethanol (β-ME), 10ng/mL murine interleukin-15 (IL-15) (PeproTech) and CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a 1:1 ratio. Cells were kept in 96 U-well plates (Greiner Bio-One) in normoxic incubators at 37°C and propagated at a density of 4x10 4 cells per well. Purity test after expansion was performed by flow cytometry to confirm exclusion of CD4 + and CD8 + T cells (typical purity > 90%).
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