Peripheral blood lymphocytes (PBLs) from independent normal human donors were isolated from purchased apheresis products (Key Biologics, Memphis, TN) using Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) density gradients40 (link),43 (link). T cells were activated by stimulating PBLs from an individual donor with 50 ng/mL anti-CD3 mAb (Miltenyi Biotec, Bergisch Gladbach, Germany), 300 IU/mL recombinant human IL-2 (rhIL-2; Novartis Pharmaceuticals, East Hanover, NJ), and 100 ng/mL recombinant human IL-15 (rhIL-15; Biological Resources Branch, National Cancer Institute, Bethesda, MD) for three days prior to transduction43 (link). Alternatively, PBLs from the same donor were cultured with 20 ng/mL recombinant human IL-7 (rhIL-7; Biological Resources Branch, National Cancer Institute) for seven days prior to transduction.
Anti cd3 mab
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD3 mAb is a monoclonal antibody that binds to the CD3 complex, a key component of the T cell receptor. This product can be used for the activation and expansion of T cells in various in vitro applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd28 mab, by Miltenyi Biotec (4 mentions)
Ficoll hypaque, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Macsprep pbmc isolation kit, by Miltenyi Biotec (2 mentions)
Recombinant human il 2 rhil 2, by Novartis (1 mentions)
Anti cd34 magnetic beads, by Miltenyi Biotec (1 mentions)
Rhil 2, by Novartis (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Jurkat e6, by American Type Culture Collection (1 mentions)
Hek293gp, by American Type Culture Collection (1 mentions)
Methyl 3h thymidine, by PerkinElmer (1 mentions)
96 well round bottom plate, by Corning (1 mentions)
Human il 7, by Miltenyi Biotec (1 mentions)
Human il 15, by Miltenyi Biotec (1 mentions)
Cxcr4, by BD (1 mentions)
Phase contrast microscopy, by Nikon (1 mentions)
Ldh activity kit, by Merck Group (1 mentions)
10 protocols using anti cd3 mab
1
Isolation and Activation of T Cells
2
Transduction and Activation of T Cells
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T cells derived from normal healthy donors were activated prior to transduction using 50 ng/mL anti-CD3 mAb (Miltenyi Biotec), 300 IU/mL rhIL-2 (rhIL-2; Novartis Pharmaceuticals), and 100 ng/mL rhIL-15 (NCI-Frederick) on day 0. T cells, Jurkat E6.1 cells, and CD8+ Jurkat E6.1 cells were transduced by spinoculation on day 3 as described elsewhere.10 (link), 31 (link) Transduced T cells or Jurkat E6.1 cells were purified by positive selection using anti-CD34 magnetic beads (Miltenyi Biotec) and maintained in complete T cell medium or Jurkat cell medium. The T cells were used in functional assays beginning on day 13.
3
Expansion of Human PBMCs with Cytokines
4
Cell Culture Protocols for Immune Cell Engineering
5
DC-Mediated T Cell Proliferation Inhibition
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Functional properties of the DCs were measured by their ability to inhibit the proliferative response of T cell in a Mixed Leukocyte Reaction (MLR) test. CD3+ T cells and monocytes/moDCs were cultured alone (as a control) or together (MLR) for 48, 72 and 96 h with a ratio of 1:10 (DC:CD3+) in ML10 medium supplemented with 10% SAB (Sigma–Aldrich, St. Louis, MO, USA) in a round bottom 96‐well plate (Corning Costar). DCs or monocytes were irradiated at 50 Gy before the culture and T cells were activated with plate‐bound anti‐CD3 mAb (1 µg/mL) (Miltenyi Biotec, Bergisch Gladbach, Germany) and anti‐CD28 mAb (0.1 µg/mL) (Clinisciences, Montrouge, France). T cells proliferation was measured after [methyl‐3H] thymidine (1 µCi/well) (PerkinElmer, Courtaboeuf, France) incubation for the last 18 h before harvesting. Radioactivity was determined using a β‐counter (1450 Trilux, Wallac, Finland). Each proliferation assay was carried out in triplicate and estimated in count per minute (cpm) and then normalized on the control condition (CD3+ T cells co‐cultured with iDC or mDC).
6
Lentiviral Transduction of Activated PBMCs
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PBMCs were activated in plates coated with 1 µg/mL anti-CD3 mAb (clone: OKT3, Miltenyi Biotec) and cultured in the presence of 3 µg/mL anti-CD28 mAb (clone: 15E8, Miltenyi Biotec), 25 U/mL human IL-7 (Miltenyi Biotec) and 50 U/mL human IL-15 (Miltenyi Biotec) for 3 days. Afterward, 8 × 104 activated human PBMCs were transduced with 0.5 µL VSV-LV (MOI 4–5) via spinfection (at 850 × g, 90 min and 32 °C) and further cultivated for 3 days.
7
Activation and Lentiviral Transduction of Human PBMCs
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Human peripheral blood mononuclear cell (PBMC) samples were obtained from healthy donors isolated by using MACSprep™ PBMC Isolation Kit (Miltenyi Biotec, Germany), according to the manufacturer’s instructions. Written informed consents were obtained from all volunteer. In brief, The PBMCs were activated for 48 h in 24-well tissue culture–treated plates (2 × 106/well) with anti-CD3 mAb (Miltenyi, Biotec, 1 μg/ml) and anti-CD28 mAb (Miltenyi, Biotec, 1 μg/ml) in a complete medium containing 90% RPMI 1640 and supplemented with 10% FBS (Gibco), 200 U/ml IL-2, 25 mM HEPPES, 55 μM 2-M, 100 U/ml penicillin, and 100 μg/ml streptomycin. After 2 days, the 2 × 105 activated T cells were infected with 250 μL of CAR-encoding lentiviral supernatant in a tissue culture–treated 24-well plate. After lentiviral infection for 24 h, the lentiviral supernatant was replaced with a fresh complete medium, and cell culture were maintained at 37°C with 5% CO2. On the 7th day after transfection, the T cells were collected for subsequent experiments.
8
PBMC Activation and Lentiviral Transduction
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Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors (n = 4) after informed consent, isolated by using MACSprep™ PBMC Isolation Kit (Miltenyi Biotec, Germany), according the manufacturer’s instructions. The PBMCs were activated for 48 h in tissue culture-treated 24-well plates (2 × 106/well) with anti-CD3 mAb (Miltenyi, Biotec, plate-bound, 1 ug/ml) and anti-CD28 mAb (Miltenyi, Biotec, soluble, 1 ug/ml) in complete medium (90% RPMI-1640 supplemented with 10% FBS (Gibco), 2 mM L-glutamine, 25 mM HEPES, 55 μM 2-M,100 U/ml penicillin, 100 μg/ml streptomycin and different cytokine combinations), and various cytokine conditions as shown in Figure 1B . After 2 days, 2 × 105 activated T cells were transfected with 500 μL the CAR-containing lentiviral supernatant in 24-well tissue culture-treated plates, and the supernatant was replaced with the fresh corresponding medium at 24 h after transfection. Culture medium change with fresh addition of cytokines was performed every other day. The T cells were transferred to 12-well tissue culture plates or 6-well tissue culture plates depending on the total cell number, and cell cultures were maintained at 37°C with 5% CO2. On the 12th day after transfection, the T cells were collected for subsequent experiments.
9
Isolation and Characterization of Fibrocytes
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Nonadherent non-T (NANT) cells were obtained, as described previously, with some modifications. 14 Briefly, PBMCs were first separated from whole blood by means of Ficoll-Hypaque (Dutscher, Brumath, France) density gradient centrifugation. After erythrocyte lysis and incubation for 1 hour at 378C, the nonadherent mononuclear cells were further depleted with an anti-CD3 mAb (Miltenyi Biotech, Paris, France).
Fibrocytes were then identified by using flow cytometry as positive for both the cell-surface marker CD45 and intracellular collagen I (see Fig E1, A-E, in this article's Online Repository at www.jacionline.org), as described previously by Moeller et al. 15 Expression of the CD34 progenitor cell marker and chemokine receptors was also assessed by means of flow cytometry with specific fluorescent antibodies directed against CD34, CCR3, CCR7, CXCR4 (BD Biosciences, San Jose, Calif), and CCR2 (R&D Systems, Lille, France). Blood fibrocyte morphology was checked by using phase-contrast microscopy (Nikon, Champigny sur Marne, France; see Fig E1,F).
Fibrocytes were then identified by using flow cytometry as positive for both the cell-surface marker CD45 and intracellular collagen I (see Fig E1, A-E, in this article's Online Repository at www.jacionline.org), as described previously by Moeller et al. 15 Expression of the CD34 progenitor cell marker and chemokine receptors was also assessed by means of flow cytometry with specific fluorescent antibodies directed against CD34, CCR3, CCR7, CXCR4 (BD Biosciences, San Jose, Calif), and CCR2 (R&D Systems, Lille, France). Blood fibrocyte morphology was checked by using phase-contrast microscopy (Nikon, Champigny sur Marne, France; see Fig E1,F).
10
CD8+ T-cell Cytotoxicity Assay
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Peripheral blood mononu clear cells (PBMC) were isolated by Ficoll density gradient centrifugation from healthy donors. Then, to isolate the CD8+T-cells from PBMCs, monocytes were depleted by plastic adherence and any cell clamps were removed using a 70 µm pre-separation lter (Miltenyi Biotec, Bergisch Gladbach, Germany). CD8+ T-cells were positively isolated from the peripheral blood lymphocytes using magnetic beads-conjugated anti-CD8 mAbs kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Subsequently,the cells were stimulated with 5 μg/ml of anti-CD3 mAb (Miltenyi Biotec, Bergisch Gladbach, Germany) and 5 μg/ml of anti-CD28 mAb (Miltenyi Biotec, Bergisch Gladbach, Germany) in the presence of 100 IU/ml recombinant human inerleukin-2. To maintain T lymphocyte expansion, half-volume medium exchange was performed every 3 days with fresh medium and recombinant human IL-2. TW03 cells were seeded at 1 × 10 3 /well in a 96-well U-bottom plate, co-cultured with CD8+ T-cells at different effector-to-target (E/T) ratio for 18 h. The cell-free supernatant was collected, and cytotoxicity was assessed using an LDH activity kit (Sigma, MAK066).
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