Peripheral venous blood was collected from subjects into syringes containing 5 U/ml heparin. Mononuclear cells were isolated by differential centrifugation (800 g, 30 min, 20°C) over Lymphoprep and washed twice with sterile phosphate-buffered saline (PBS; GIBCO, Paisley, UK) at 500 g (5 min, 20°C). Cells were resuspended in 10 ml RPMI-1640 medium (Invitrogen) supplemented with 100 U/ml of penicillin (GIBCO), 100 µg/ml streptomycin (GIBCO) and 20 mM HEPES pH 7.4 (Sigma-Aldrich), and plated at a density of approximately 5×106 cells/ml in 8 cm2 Nunclon™ Surface tissue culture dishes (Nunc, Roskilde, Denmark) at 37°C, 5% CO2. After 2 h, non-adherent cells were discarded and 10 ml of fresh RPMI supplemented with 10% foetal bovine serum (FBS; Sigma) added to each tissue culture dish. Cells were then cultured for 5 days at 37°C, 5% CO2, with the addition of a further 10 ml of fresh 10% FBS/RPMI after 24 h. Cells were then washed twice in PBS, scraped and spun down at 500 g (5 min, 20°C). Cells were resuspended into X-vivo-15 medium (Cambrex, MD, USA) and plated at a density of 106 cells per 8 cm2 Nunclon™ dish for a further 25 h at 37°C, 5% CO2.
X vivo 15 medium
Manufactured by Cambrex
Sourced in United States
X-VIVO 15 medium is a serum-free and protein-free cell culture medium designed for the growth and maintenance of a variety of cell types, including primary cells, stem cells, and immortalized cell lines. It provides a defined, chemically-formulated environment for cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Nunclon surface tissue culture dishes, by Thermo Fisher Scientific (2 mentions)
Hepes ph 7, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Lymphoprep, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Recombinant human il 4, by R&D Systems (1 mentions)
Sodium heparin tube, by BD (1 mentions)
Facs caliber cytometer, by BD (1 mentions)
E coli lps, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Rtnf α, by R&D Systems (1 mentions)
Rgm csf, by R&D Systems (1 mentions)
Sterile pbs, by Thermo Fisher Scientific (1 mentions)
7 protocols using x vivo 15 medium
1
Isolation and Culture of Mononuclear Cells
2
Generating CD8+ T cells for Gp100 Immunotherapy
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Leukocytes were obtained from buffy coats of healthy individuals and purified using Ficoll density centrifugation. Monocyte-derived DCs were obtained from leukocytes as reported elsewhere [36 (link)]. Cells were cultured in X-VIVO 15 medium (Cambrex, Wiesbaden, Germany) supplemented with 2% human serum. CD8+ T cells transfected with a TCR recognizing the gp100 (280–288) epitope were generated as described previously [39 (link)]. Blood cells from controls were obtained from buffy coats from anonymous healthy donors of the blood bank after informed consent. Experiments were approved and carried out in accordance with the guidelines and regulations of the Radboud University Medical Centre, Nijmegen, The Netherlands.
3
Monocyte-Derived Dendritic Cell Generation
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Monocytes were derived from peripheral blood obtained from four healthy volunteers after they signed an informed consent. Peripheral blood cells were diluted 1:3 with phosphate-buffered saline. The suspension was transferred to a 15-mL conical tube containing 5 mL of Ficoll-Paque 1.077 density (GE Healthcare, UK) and centrifuged (30 min/500 g) at 22°C. The cells from the interface were collected, ressuspended, and centrifuged again (5 min/500 g). Cells were then separated according to CD14 expression using a magnetic selection column (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD14 cell population was dispensed into six-well plates containing X-vivo 15 medium (Cambrex) supplemented with antibiotic-antimycotic solution (Gibco). To generate immature dendritic cells (iDCs), the cells were cultured in the presence of human recombinant IL-4 (20 ng/mL) and GM-CSF (50 ng/mL) (both from R&D Systems, Minneapolis, MN, USA) for 6 days (20 (link)). Mature DCs were obtained after iDC stimulation with LPS 100 ng/mL for 24 h (Sigma-Aldrich). Monocytes and monocyte-derived immature and mature DCs were analyzed for chemokines receptor as described in the following section.
4
Bovine PBMC Isolation and Stimulation
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Whole blood was drawn from Holstein bull calves (<2 years old) into sodium heparin tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Histopaque 1077 (Sigma-Aldrich, St Louis, MO, USA) was used to separate the PBMCs from whole blood, as per the manufacturer's instructions, as previously described.26 (link)
Cell preparations were maintained in X-VIVO 15 medium (Cambrex) at 1 × 106 cells/ml at 37°C, 5% CO2 in the presence of known or test agonists, or medium and/or vehicle only as controls for 24–48 h. Samples from a minimum of three calves were compared. Bovine PBMCs were cultured with lipo-CRX, E. coli LPS (Sigma) or MPL or CRX-527 (Invivogen) at the indicated concentrations along with media and vehicle only controls. Vehicle is the same liposome preparation, but without added CRX-527 incorporated within the liposome membrane. Following incubation, the supernatant fluids were collected and cells were stained for γδ TCR (clone GD3.8) and IL-2R (clone LCBT2A, VMRD) as previously described.21 (link)24 (link, link, link) and assessed on a BD FACS-Caliber cytometer.
Cell preparations were maintained in X-VIVO 15 medium (Cambrex) at 1 × 106 cells/ml at 37°C, 5% CO2 in the presence of known or test agonists, or medium and/or vehicle only as controls for 24–48 h. Samples from a minimum of three calves were compared. Bovine PBMCs were cultured with lipo-CRX, E. coli LPS (Sigma) or MPL or CRX-527 (Invivogen) at the indicated concentrations along with media and vehicle only controls. Vehicle is the same liposome preparation, but without added CRX-527 incorporated within the liposome membrane. Following incubation, the supernatant fluids were collected and cells were stained for γδ TCR (clone GD3.8) and IL-2R (clone LCBT2A, VMRD) as previously described.21 (link)
5
Dendritic Cell-Mediated Lymphocyte Proliferation
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Dendritic cells were derived from monocytes selected for CD14 by magnetic selection column (Myltenyi Biotec, Bergisch Gladbach, Germany). The CD14+ cell population was dispensed into six-well plates containing X-vivo 15 medium (Cambrex) supplemented with antibiotic-antimycotics (Gibco). To generate immature dendritic cells (DCs), the cells were cultured in the presence of 20 ng/mL recombinant IL-4 (rIL-4) and 50 ng/ml rGM-CSF (both from R&D Systems, Minneapolis, MN) for six days (indicated as D6 cells). Mature DCs were obtained after 24 h (D7) after stimulation of the immature DC culture with 10 ng/mL rTNF-α (R&D System) plus 0.01 mmol/L of PGE2 (Sigma) [23] (link).
In order to set up the MLR, matured DCs were irradiated at 1,500 rads and co-cultured with allogeneic lymphocytes at the concentration of 1 DC:100 Lymphocytes (1×104:1×106 respectively) in presence or absence of 1×105 allogeneic hMSCs. The proliferation rates were determined after four days of co-culture. Lymphocyte proliferation was measured by KI-67 nuclear staining on CD3+ gated population. The lymphocytes were gated based on SSC vs FSC, followed by CD3 gating; the selected events were analyzed in a second plot for KI-67 expression. The proliferation negative control was lymphocytes without allogeneic DCs stimulation.
In order to set up the MLR, matured DCs were irradiated at 1,500 rads and co-cultured with allogeneic lymphocytes at the concentration of 1 DC:100 Lymphocytes (1×104:1×106 respectively) in presence or absence of 1×105 allogeneic hMSCs. The proliferation rates were determined after four days of co-culture. Lymphocyte proliferation was measured by KI-67 nuclear staining on CD3+ gated population. The lymphocytes were gated based on SSC vs FSC, followed by CD3 gating; the selected events were analyzed in a second plot for KI-67 expression. The proliferation negative control was lymphocytes without allogeneic DCs stimulation.
6
Isolation and Culture of Monocyte-Derived Macrophages
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Peripheral venous blood samples were collected into heparin (5 U/ml). Mononuclear cells were isolated by differential centrifugation (900 g , 30 min, 20°) over Lymphoprep® (Axis-Shield, Oslo, Norway) and washed twice with sterile PBS (GIBCO, Paisley, UK) at 300 g (5 min, 20°). Cells were resuspended in 10 ml RPMI-1640 (Invitrogen, Paisley, UK) supplemented with 100 U/ml of penicillin (GIBCO) and 100 μg/ml streptomycin (GIBCO) and 20 mm HEPES buffer (Sigma-Aldrich, Poole, UK) (RPMI), and plated at a density of approximately 5 × 106 cells/ml in 8 cm2 Nunclon™ Surface tissue culture dishes (Nunc, Roskilde, Denmark). After an initial culture period of 2 hr at 37°, 5% CO2, the non-adherent cells were discarded and 10 ml of fresh RPMI supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) (10% FBS/RPMI) was added to each tissue culture dish. Cells were then cultured for 5 days at 37°, 5% CO2, with the addition of a further 10 ml fresh 10% FBS/RPMI after 24 hr. Adherent cells were scraped on day 5 and re-plated at 105/well for cytokine analysis and 106/well for RNA extraction in X-Vivo-15 medium (Cambrex, Walkersville, MD). These primary MDM were incubated overnight at 37°, 5% CO2 to adhere, and then stimulated for 4 hr (transcriptomic studies) or 24 hr (cytokine studies) with (100 : 1) heat-killed Escherichia coli clone NCTC 10418 (HkEc).
7
Lentiviral CAR-T Cell Production
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A third generation self-inactivating lentiviral configuration encoding the 4g7 CAR19 (CD19scFv-41BB-CD3), HIV central polypurine tract (CPPT), Rev Responsive Element (RRE), and mutated Woodchuck post regulatory element (WPRE) was manufactured and released as previously described (24, 25) . Healthy donor peripheral blood mononuclear cells (PBMNC) were supplied cryopreserved (HemaCare) and were thawed and then activated with anti-CD3, anti-CD28 Expact beads (Miltenyi Biotec) in X-Vivo 15 medium (Cambrex) supplemented 5% human AB serum and 100 ng/ml interleukin-2 (IL2) (Miltenyi Biotec). After a single round of lentiviral transduction, the beads were removed by CliniMacs magnetic depletion, and the cells were subjected to multiplex gene editing by electroporation of high grade TALEN mRNA (Eufets) targeting TRAC and CD52. Cells were suspended in BTXpress cytoporation media-T in a customised GMP compliant electroporation chamber for use with an Agile Pulse device (BTX Harvard Apparatus). Thereafter, cells were transferred to a Wave Bioreactor 2/10 (GE Healthcare) and expanded for up to ten days before being depleted of residual TCR expressing cells by CliniMACS TCR depletion (Miltenyi Biotec). The resulting cell product was cryopreserved in dose aliquots of 50 million cells.
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