A 6-well plate was coated with 1 μg/mL of anti-CD3 (clone OKT3) and anti-CD28 (clone CD28.2) antibodies at 4 °C, overnight, 1 day before blood collection. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors. The use of human PBMCs was approved by the Ethics Committee of Lanzhou University Second Hospital, and all donors gave informed consent. The day of blood collection was signed as d0. After 4-hour culture, the suspension cells were transferred to the 6-well plate already coated with CD3 and CD28 antibodies. Then, a new 6-well plate was coated by 50 μg/mL of RetroNectin (Takara, Japan) at 4 °C, overnight, on d0. After 18-hour stimulation by CD3/CD28 antibodies, the suspension cells were transferred into the RetroNectin coated 6-well plate and infected by lentivirus at MOI = 10, 5 μg/mL of polybrene was used as an infection promoting agent. The day of lentivirus infection was marked as d1. CAR-T cells were cultured in X–VIVO 20 Serum-free Medium (Lonza, Switzerland) containing 200 U/mL of Recombinant Human IL-2 (PeproTech, USA). The cell density was kept at 1 × 106/mL, the medium was changed every 3 days.
X vivo 20 serum free medium
Manufactured by Lonza
Sourced in Switzerland
X-vivo 20 is a serum-free medium developed for the culture of a variety of cell types, including hematopoietic and adherent cells. It is a chemically defined, protein-free formulation that supports the growth and expansion of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Merck Group (4 mentions)
L glutamine, by Merck Group (4 mentions)
Interleukin 6 (il 6), by Roche (4 mentions)
Il 1β, by Roche (3 mentions)
Tgf β, by Roche (3 mentions)
X vivo 20 medium, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Anti ifn γ, by Roche (2 mentions)
Anti il 4, by Roche (2 mentions)
Dynal cd4 positive isolation kit, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Retronectin, by Takara Bio (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Duoset elisa kit, by R&D Systems (1 mentions)
Cd28 antibody, by BD (1 mentions)
Human il 13 elisa kit, by Thermo Fisher Scientific (1 mentions)
Anti cd3 antibody, by BD (1 mentions)
Easysep human cd4 t cell isolation kit, by STEMCELL (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
8 protocols using x vivo 20 serum free medium
1
Lentiviral CAR-T Cell Production
2
Activating and Profiling CD4+ T Cells
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CD4+ T lymphocytes were isolated from frozen PBMC using the EasySep™ Human CD4+ T Cell Isolation Kit (Stem Cell Technologies, Vancouver, BC, Canada). CD4+ cells were resuspended at a concentration of 1 × 106 cells/mL in X-VIVO 20 serum-free medium (Lonza Group Ltd., Basel, Switzerland) supplemented with 1% penicillin/streptomycin. CD4+ cells (500,000 cells/well) were incubated in 48-well plates previously coated with an anti-CD3 antibody (Becton Dickinson, Franklin Lakes, NJ, USA) with or without a CD28 antibody (Becton Dickinson, Franklin Lakes, NJ, USA), which induces cell activation. After 6 h of incubation at 37 °C with 5% CO2, the supernatant and cell pellets were collected for further analysis. The cytokines IL-17, IL-22, TNF-α, TFG-β, and IL-1β were measured in the culture supernatant by DuoSet® ELISA Kits (R&D Systems, Minneapolis, MN, USA). For the IL-13 measurement, a human IL-13 ELISA kit was used (Invitrogen, Thermo Fisher, USA). IL-21 interleukin was measured in the cell pellet by western blot.
3
Cryopreservation of Cell Suspensions
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After isolation the cells were suspended in X-vivo 20 serum free medium (Lonza; Walkersville, Maryland, USA). Cells frozen in X-vivo were supplemented with ProFreeze chemically defined freeze medium (Lonza; Verviers, Belgium). Cell suspensions were mixed with DMSO (Sigma Aldrich) to prevent damage and then cryopreserved in liquid nitrogen.
4
Th17 Cell Differentiation Protocol
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CD4+ T cells were activated with plate-bound
α-CD3 (3.75 μg/mL; Immunotech) and soluble α-CD28
(1 μg/mL; Immunotech) in X-VIVO 20 serum-free medium (Lonza).
X-VIVO 20 medium was supplemented withl -glutamine (2 mM,
Sigma-Aldrich) and antibiotics (50 U/mL penicillin and 50 μg/mL
streptomycin; Sigma-Aldrich). Th17 cell differentiation was induced
using a cytokine cocktail of IL-6 (20 ng/mL; Roche), IL-1β (10
ng/mL), and TGF-β (10 ng/mL) in the presence of the neutralizing
Ab anti-IFN-γ (1 μg/mL) and anti-IL-4 (1 μg/mL)
to block Th1 and Th2 polarization, respectively. For the control cells
(Th0), CD4+ T cells were TCR-stimulated with α-CD3
and α-CD28 in the presence of neutralizing Ab. All cytokines
and neutralizing Ab were purchased from R&D Systems, unless otherwise
stated. All cultures were maintained at 37 °C in a humidified
atmosphere of 5% (v/v) CO2/air.
α-CD3 (3.75 μg/mL; Immunotech) and soluble α-CD28
(1 μg/mL; Immunotech) in X-VIVO 20 serum-free medium (Lonza).
X-VIVO 20 medium was supplemented with
Sigma-Aldrich) and antibiotics (50 U/mL penicillin and 50 μg/mL
streptomycin; Sigma-Aldrich). Th17 cell differentiation was induced
using a cytokine cocktail of IL-6 (20 ng/mL; Roche), IL-1β (10
ng/mL), and TGF-β (10 ng/mL) in the presence of the neutralizing
Ab anti-IFN-γ (1 μg/mL) and anti-IL-4 (1 μg/mL)
to block Th1 and Th2 polarization, respectively. For the control cells
(Th0), CD4+ T cells were TCR-stimulated with α-CD3
and α-CD28 in the presence of neutralizing Ab. All cytokines
and neutralizing Ab were purchased from R&D Systems, unless otherwise
stated. All cultures were maintained at 37 °C in a humidified
atmosphere of 5% (v/v) CO2/air.
5
Purification and Th17 Polarization of CD4+ T Cells
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Human mononuclear cells were isolated from the umbilical cord blood of healthy neonates (Turku University Central Hospital, Turku, Finland) using Ficoll-Paque isolation (Ficoll-Paque PLUS; GE Healthcare). CD4+ cells were further purified (Dynal CD4 Positive Isolation Kit; Invitrogen) and after the isolation cells from several individuals were pooled. Cells were activated with plate-bound αCD3 (750 ng/24-well culture plate well; Immunotech) and soluble αCD28 (1 μg/mL; Immunotech) in a density of 0.5 × 106 cells/mL of X-vivo 20 serum-free medium (Lonza). The media was supplemented with 2 mM L-glutamine (Sigma-Aldrich), and 50 U/mL penicillin and 50 μg/mL streptomycin (Sigma-Aldrich). Cells were polarized toward Th17 direction with IL6 (20 ng/mL; Roche), IL1β (10 ng/mL) and TGFβ (10 ng/mL) in the presence of neutralizing anti-IFNγ (1 μg/mL) and anti-IL4 (1 μg/mL). Cells activated without differentiating cytokines but with only neutralizing antibodies were also cultured as controls (Th0). All cytokines and neutralizing antibodies were from R&D Systems unless otherwise stated.
6
Induced Th17 Cell Differentiation
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CD4+ T cells were activated with plate-bound α-CD3 (3.75 µg/ml; Immunotech) and soluble α-CD28 (1 μg/mL; Immunotech) in X-vivo 20 serum-free medium (Lonza). X-vivo 20 medium was supplemented with L-glutamine (2 mM, Sigma-Aldrich) and antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin; Sigma-Aldrich). Th17-cell differentiation was induced using a cytokine cocktail of IL-6 (20 ng/mL; Roche), IL-1β (10 ng/mL) and TGF-β (10 ng/mL) in the presence of the neutralizing antibodies anti-IFN-γ (1 μg/mL) and anti-IL-4
(1 μg/mL) to block Th1 and Th2 polarization, respectively. For the control cells (Th0), CD4+ T cells were TCR stimulated with α-CD3 and α-CD28 in the presence of neutralizing antibodies. All cytokines and neutralizing antibodies were purchased from R&D Systems, unless otherwise stated. All cultures were maintained at 37°C in a humidified atmosphere of 5% (v/v) CO 2 /air.
(1 μg/mL) to block Th1 and Th2 polarization, respectively. For the control cells (Th0), CD4+ T cells were TCR stimulated with α-CD3 and α-CD28 in the presence of neutralizing antibodies. All cytokines and neutralizing antibodies were purchased from R&D Systems, unless otherwise stated. All cultures were maintained at 37°C in a humidified atmosphere of 5% (v/v) CO 2 /air.
7
Isolation and Polarization of Th17 Cells
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Human cord blood mononuclear cells (CBMCs) were isolated from the umbilical cord blood of healthy neonates (Turku University Central Hospital, Turku, Finland) using the Ficoll-Paque density gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare). Naive CD4 + T cells were further purified using CD4 + Dynal positive selection beads (Dynal CD4 Positive Isolation Kit; Invitrogen). CD4 + T-cells were stimulated with plate-bound α-CD3 (3.75 µg/ml; Immunotech) and soluble α-CD28 (1 μg/mL; Immunotech) in X-vivo 20 serum-free medium (Lonza). X-vivo 20 medium was supplemented with L-glutamine (2 mM, Sigma-Aldrich) and antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin; Sigma-Aldrich). Th17 cell polarization was induced using a cytokine cocktail of IL-6 (20 ng/mL; Roche), IL-1β (10 ng/mL) and TGF-β (10 ng/mL) in the presence of neutralizing anti-IFN-γ (1 μg/mL) and anti-IL-4 (1 μg/mL) to block Th1 and Th2 differentiation, respectively. For the control cells (Th0), CD4 + T-cells were TCR stimulated with α-CD3 and α-CD28 in the presence of neutralizing antibodies (without differentiating cytokines). All cytokines and neutralizing antibodies used in the study were purchased from R&D Systems unless otherwise stated. All cultures were maintained at 37°C in a humidified atmosphere of 5% (v/v) CO 2 /air.
8
Antigen-specific Treg Suppression Assay
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Treg function was studied by antigen-specific suppression assay as described earlier. 33 Briefly, effector T cells were generated by immunizing mice with purified AHC protein. CD4-positive cells from spleen were purified using T cell isolation kit (Miltenyi Biotech, Teterow, Germany) and labeled with 10 μmol/L 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma Chemicals, St. Louis, MO, USA) and used as effector cells. Spleen cells from mice orally dosed with recombinant M. smegmatis secreting AHC protein and regulatory T cells were isolated using the CD4 + CD25 + regulatory T cell isolation kit (Miltenyi Biotech) and labeled with 6 μmol/L PKH26 (Sigma Chemicals) to discriminate the effector and regulatory CD4 + population. Effector T cells (1×10 5 ) and regulatory cells were taken in equal ratios and activated with 10 μg/mL of antigen (purified AHC protein) in X vivo 20 serumfree medium (Lonza, Basel, Switerland). After 5 days of incubation, cells were stained with CD4-APC (eBiosciences, San Diego, CA, USA). 34 Proliferation of CD4 + effector cells was measured by CFSE dilution using FACS CANTO II (Becton Dickinson) and analyzed using FlowJO software (Tree star). The proliferation index of T cells was calculated from the FlowJO software.
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