For T cell activation, total PBMC samples from healthy donors (ALLCELLS, Alameda, CA) were enriched for CD3+ T cell populations using CD3 MicroBeads (Miltenyi Biotec, Auburn, CA, cat # 130-050-101) according to manufacturer’s instructions. Cells were then maintained in OpTmizer CTS T Cell expansion SFM plus T cell expansion supplement (Gibco, Grand Island, NY, cat # A1048501) complete with 5% CTS Immune Cell SR (Gibco, cat # A2596101), 2 mM Glutamine (Gibco, cat # 35050-061) and P/S solution. T cells were activated using Dynabeads Human T-Activator CD3/CD28 beads (Gibco, cat # 11131D). Surface FACS using anti-CD69 (Biolegend, London, UK, cat # 310904) staining was used to detect and confirm the activated state of T cells. TGFβ neutralizing antibody was added to cells within 24 h, and after 72 h incubation, cells were washed and stained with anti-CD4 (BD Biosciences, cat # 560768) and anti-CD8a (eBioscience, Grand Island, NY, cat # 56-0086-82) antibodies and subsequently analyzed with flow cytometry.
Cts immune cell sr
Manufactured by Thermo Fisher Scientific
Sourced in Australia
The CTS Immune Cell SR is a laboratory equipment product designed for the processing and culture of immune cells. It provides a controlled environment for the maintenance and expansion of various types of immune cells, such as T cells, B cells, and natural killer cells. The CTS Immune Cell SR is intended to support research and development activities in the field of immunology and cell therapy.
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Lab products found in correlation
Interleukin 2 (il 2), by Thermo Fisher Scientific (4 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (3 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (2 mentions)
X vivo 15 media, by Lonza (2 mentions)
Human t activator cd3 cd28, by Thermo Fisher Scientific (2 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Anti cd69, by BioLegend (1 mentions)
Anti cd8a, by Thermo Fisher Scientific (1 mentions)
Dynabeads human t activator cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions)
Anti cd4, by BD (1 mentions)
Cd3 microbeads, by Miltenyi Biotec (1 mentions)
Human t cell transact, by Miltenyi Biotec (1 mentions)
Human t cell isolation kit, by STEMCELL (1 mentions)
Recombinant human il 7 and il 15, by Miltenyi Biotec (1 mentions)
T cell transact, by Miltenyi Biotec (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by American Type Culture Collection (1 mentions)
Cts optmizer t cell expansion sfm, by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
9 protocols using cts immune cell sr
1
T Cell Activation and Analysis
2
Expansion of Potent Natural Killer Cells
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Sufficiently activated mature NK cells (super NK [SNK]) were expanded from healthy donor PBMC using a DCTY NK serum‐free medium culture kit (LY1201; Beijing DCTY Biotech Co., Ltd). During the cell culture, the medium was supplemented with 8% CTS‐immune cell SR (Gibco). In brief, the culture flask was coated with NK reagent A at 4°C for 24 h. The coating fluid was discarded, and the PBMCs were inoculated with a medium containing reagent B. In the first week of cell culture, the medium was added to the cells with NK reagent C. Subsequently, the cells were cultured in a medium containing NK reagent D. The ex vivo SNK cells were continuously cultured until Days 18–21, and the SNK cells with a purity of exceeding 90% were used for further in vitro experiments.
3
Cryopreserved PBMC Culture and TNBC Cell Lines
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Cryopreserved human PBMCs were acquired from ALLCELLS (# PB006F). PBMCs were cultured in CTS OpTmizer media (obtained from Gibco, # A1048501), containing IL-2 (Miltenyi Biotec, # 130-097-748), human serum AB (Seralab, # GEM-100-318), and CTS Immune Cell SR (Gibco, # A2596101). Human T Cell TransAct (Miltenyi Biotec t# 130-111-160) was used to activate T cells. PBMCs were cryopreserved in 90% albumin/10% DMSO.
HCC70-GFP were engineered from HCC70 cells (ATCC, # CRL-2315) using an in house rLV encoding NanoLuc_T2A_EGFP construct and AMSbio (# LVP323-PBS), respectively, using the manufacturer’s protocols. TNBC patient-derived CAFs were obtained from BioIVT (cancer fibroblasts #233831P1). All cell lines and CAFs were maintained in DMEM supplemented with 10% heat-inactivated FBS in 5% CO2 at 37°C.
HCC70-GFP were engineered from HCC70 cells (ATCC, # CRL-2315) using an in house rLV encoding NanoLuc_T2A_EGFP construct and AMSbio (# LVP323-PBS), respectively, using the manufacturer’s protocols. TNBC patient-derived CAFs were obtained from BioIVT (cancer fibroblasts #233831P1). All cell lines and CAFs were maintained in DMEM supplemented with 10% heat-inactivated FBS in 5% CO2 at 37°C.
4
Human PBMC Isolation and T Cell Expansion
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Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors after informed consent and were used in experiments under a Human Research Ethics Committee (HREC) approved protocol (Sydney Children’s Hospitals Network, LNR/13/SCHN/241). We isolated PBMCs by separation over Lymphoprep, and then enriched for T cell populations using a human T cell isolation kit according to the manufacturer’s instructions (Stem Cell technologies, Tullamarine, Australia). T cells were activated with T Cell TransAct (Miltenyi Biotec Australia, Sydney, Australia) in CTS OpTmizer T cell expansion medium supplemented with CTS Immune cell SR (2.5%, ThermoFisher Scientific Australia) and recombinant human IL-7 and IL-15 (10 ng/mL and 5 ng/mL respectively, Miltenyi Biotech Australia). Human T cells were subsequently expanded in the above medium supplemented with IL-7 and IL-15 every 48 hr for ~12 days prior to use in experiments.
5
Expansion and Activation of Primary Human T Cells
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Primary human T cells were cultured in OpTmizer CTS T-cell Expansion SFM (ThermoFisher, Waltham, MA) containing 2.5% CTS Immune Cell SR (ThermoFisher), L-Glutamine, Penicillin/Streptomycin, N-Acetyl-L-cysteine (10 mM, Sigma–Aldrich, St. Louis, MO), IL-2 (300 IU/mL, PeproTech, Rocky Hill, NJ), IL-7 (5 ng/mL, PeproTech), and IL-15 (5 ng/mL, PeproTech) at 37 °C and 5% CO2. Prior to electroporation T cells were activated with Dynabeads Human T-Activator CD3/CD28 (ThermoFisher) at a 2:1 bead:cell ratio for 48 h. Following electroporation, T cells were maintained at 1 × 106 cells/mL in a 24-well or 12-well plate. K562 cells (ATCC CCL-243) were cultured at a density of 5 × 105 cells per mL in RPMI with 10% FBS and 1× Penicillin/Streptomycin. Cells were kept at 37 °C and 5% CO2.
6
Cryopreserved PBMC CD22-CAR T-cell Generation
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Cryopreserved human PBMCs (ALLCELLS) were thawed and plated at a density of 1 × 106 cells/ml in X-vivo-15 media (Lonza) supplemented with 5% human AB serum (Gemini) or CTS Immune Cell SR (Thermo Fisher Scientific) and 20 ng/ml IL-2 (Miltenyi Biotech) for an overnight culture at 37°C. The next day, the PBMCs were activated using human T activator CD3/CD28 (Life Technology) in serum-free X-vivo-15 media without IL-2. One million activated PBMCs (in 600 μl) were immediately incubated without removing the beads in an untreated 12-well plate pre-coated with 30 μg/ml Retronectin (Takara) in the presence of lentiviral particles encoding the CD22 targeting CAR for 2 h at 37°C. Six hundred microliters of 2× X-vivo-15 media (X-vivo-15, 10% human AB serum and 40 ng/ml IL-2) was added after 2 to 3 h, and the cells were incubated at 37°C for 72 h.
7
Expansion and Electroporation of T Cells
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T cells were cultured in OpTmizer CTS T cell Expansion SFM containing 2.5% CTS Immune Cell SR (ThermoFisher, Waltham, MA), l -Glutamine, Penicillin/Streptomycin, N-Acetyl-l -cysteine (10 mM), IL-2 (300 IU/mL), IL-7 (5 ng/mL), and IL-15 (5 ng/mL) at 37 °C and 5% CO2. T cells were activated with Dynabeads Human T-Activator CD3/CD28 (ThermoFisher, Waltham, MA) at a 2:1 bead:cell ratio for 48–72 h prior to electroporation. Following electroporation, T cells were maintained at ~1 × 106/mL in normal tissue culture flasks for experiments optimizing editing efficiency. For large-scale expansion studies and functional assays, edited T cells were expanded in gas-permeable rapid expansion (G-Rex) vessels (Wilson Wolf Manufacturing, New Brighton MN).
8
SWIFF-CAR T Cell Engineering Protocol
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Human PBMCs () were thawed and plated at 1 × 106 cells/ml in X-vivo-15 media (Lonza) supplemented with 5% hAB serum (Gemini) or CTS Immune Cell SR (ThermoFisher) and 20 ng/ml IL-2 (Miltenyi Biotech) for overnight culture at 37 °C. The next day, the PBMCs were activated using human T activator CD3/CD28 (Life Technology) in serum-free X-vivo-15 media without IL-2. One million activated PBMCs (in 600 μl) were immediately incubated without removing the beads in an untreated 12-well plate pre-coated with 30 μg/ml retronectine (Takara) in the presence of lentiviral particles encoding the engineered SWIFF-CAR for 2 h at 37 °C. Six hundred microliters of 2x X-vivo-15 media (X-vivo-15, 10% hAB serum and 40 ng/ml IL-2) was added after 2 to 3 h, and the cells were incubated at 37 °C for 72 h. If required, transduced T-cells were then expanded for 11 days in G-Rex10 (Wilson Wolf) in 40 ml of complete X-vivo-15 media.
9
Activating NK Cells for Immunotherapy
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NK cells were cultured in CTS AIM VTM (ThermoFisher, Waltham, MA) with 5% CTS Immune cell SR (ThermoFisher, Waltham, MA), and 100 IU/mL IL-2 (PeproTech, Rocky Hill, NJ). NK cells were activated by co-culture with X-irradiated (100 Gray) feeder cells (K562 expressing membrane-bound IL-21 and 41BB-L) at various feeder to NK ratios (2:1 prior to electroporation, 5:1 72 hours after Neon electroporation, or 1:1 immediately after MaxCyte electroporation).
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