To measure cytokine secretion, magnetic-activated cell sorting–purified CD4+ T cells (Miltenyi) from healthy donors were stimulated with anti-human plate-bound CD3 (1.0 μg/ml; clone OKT3, eBioscience) and anti-human soluble CD28 (0.5 μg/ml; clone CD28.2, eBioscience). GT13072 or GT14829 was added at the beginning of the culture. After 48 hours, the culture supernatants were taken for cytokine analysis. Cytokines were measured using the Meso Scale Discovery (MSD) platform according to the manufacturer’s instructions.
Clone cd28
Manufactured by Thermo Fisher Scientific
Sourced in United States
Clone CD28.2 is a mouse monoclonal antibody that recognizes the human CD28 cell surface antigen. CD28 is a co-stimulatory molecule expressed on the surface of T cells that plays a critical role in T cell activation and function.
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Lab products found in correlation
Clone okt3, by Thermo Fisher Scientific (7 mentions)
Anti cd28, by Thermo Fisher Scientific (3 mentions)
Puregene cell kit, by Qiagen (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Clone ucht1, by Thermo Fisher Scientific (2 mentions)
Anti cd3, by Thermo Fisher Scientific (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Cd4 t cells, by Miltenyi Biotec (1 mentions)
Meso scale discovery (msd), by Mesoscale (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Anti human cd3, by Thermo Fisher Scientific (1 mentions)
Clone 37, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
T cell isolation kit, by Miltenyi Biotec (1 mentions)
Mouse regulatory t cell staining kit, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse tgf β, by R&D Systems (1 mentions)
Recombinant human il 2, by R&D Systems (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
16 protocols using clone cd28
1
Cytokine Secretion Assay for CD4+ T Cells
2
Expansion and Purification of CD3+ T Cells from AML Patients
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CD3+ cells were isolated from peripheral blood (PB) AML patient samples using EasySep (Stem Cell Technologies) and re-suspended at a concentration of 1×107cells/2 mL in RPM1 + 10% FBS-HI + rhIL-2 (250 IU/mL, Proleukin, Chiron) + anti-CD28 antibody (5 ug/mL, clone CD28.2, eBioscience). Cells were then added to one well of a 24-well plate that had been pre-coated for 2 hours with anti-CD3 antibody (Clone OKT3, eBioscience) and cultured for 4 days at 37°C with 5% CO2. Cells were harvested on day 4, resuspended in fresh RPMI + 10% FBS-HI + rhIL2 (250 IU/ml) and replated into one well of a 6-well plate. Cells were further cultured and expanded for 14–20 days, feeding with fresh full medium containing rhIL-2 (250 IU/ml) every 3–4 days. At the end of T cell expansion, the purity of CD3+ T cells was checked by flow cytometry. DNA from the cultured T cells was extracted by PureGene Cell kit (Qiagen).
3
CD4+ and CD8+ T Cell Expansion
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CD4+ and CD8+ T cells were separately isolated from PBMCs using MACS (Miltenyi, according to the manufacturer’s instructions). CD4+ or CD8+ T cells (100,000 per well) were plated on anti-CD3-coated (2 μg/ml; clone OKT3; eBioscience) 96-well U-bottom plates in RPMIfs supplemented with anti-CD28 (2 μg/ml; clone CD28.2; eBioscience) and IL-2 (60 IU/ml; Peprotec) and treated with 5 μM of oligonucleotides for a total treatment time of six days without the use of a transfection reagent. Activation medium and oligonucleotides were replaced after three days. As mock control, cells were cultivated in activation medium without oligonucleotide. On day six after start of treatment, cells were transferred to uncoated 96-well U-bottom plates and cultivated in cell culture medium supplemented with IL-2 (20 IU/ml) in the absence of oligonucleotides. Cells were split 1:2 every third day. hCD39 protein expression was analyzed on day three, six and eleven after removal of oligonucleotides by flow cytometry.
4
PBMC Proliferation Assay with Stimulation
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On day 0, PBMC from each donor were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, Oregon) and seeded on each surface in the presence or absence of human anti-CD3 (1 μg/ml, clone OKT3) and anti-CD28 (3 μg/ml, clone CD28.2) (eBioscience) monoclonal antibodies (mAbs) that were used as polyclonal stimuli. On day 4, proliferation of CFSE-labeled cells was assessed by flow cytometry.
5
Activating Cord Blood T Cells
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Cord blood T cells were matured using an anti-CD3 and anti-CD28 co-stimulation method. Briefly, anti-CD3 antibodies (OKT3, Abcam, Cambridge, UK) were immobilised in 24 well plates by adding 2.5 μg/mL in Hank’s Balanced Salt Solution (HBSS) into each well, and refrigerating overnight or incubated for 3 h at 37 °C, and then washed with HBSS. At initiation of culture, anti-CD28 antibodies (Clone CD28.2, eBiosciences, San Diego, CA, USA) were added to a final concentration of 1 µg/mL with 1 × 106 CBTC to each well in a total volume of 1 mL. After three days of culture, cells were counted and reseeded at 1 × 106/mL with the addition of rhIL-2 (10 ng/mL), and this process was repeated on day 5. On day 7 the CD45RA/RO surface expression was measured by flow cytometry.
6
TCR-signaling Ability Assays with Leukemic Cells
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TCR-signaling ability assays were performed with 2×107 wild-type or leukemic cells resuspended in RPMI medium (200 μL) and stimulated for 2 minutes (min) at 37°C with avidin (14 μg) and biotinylated anti-CD3 (10 μg; clone 2C11, BD Pharmingen, for mouse cells and clone UCHT1, eBiosciences for human cells) and biotinylated anti-CD28 (10 μg; clone 37.51, BD Pharmingen, for mouse cells and clone CD28.2, eBiosciences for human cells). Unstimulated (control) cells were incubated with avidin alone. After lysis in 2X TNE buffer (100 mM Tris, 2% Nonidet P-40, 40 mM EDTA) supplemented with protease and phosphatase inhibitors, protein extracts (approx. 60 μg) were analyzed by immunoblot (see Online Supplementary Methods and Online Supplementary Table S2). P-Tyr levels were quantified on the entire lane and normalized to ACTIN. P-AKT protein levels were normalized to AKT.
7
Isolation and Stimulation of CD4+ T Cells
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CD4 T cells were isolated from PBMCs retrieved from liquid nitrogen using the EasySep™ Human Isolation Kit (Stem Cell Technologies) according to the manufacturer’s protocol. The isolated cells were assessed for purity using flow cytometry. Briefly, cells were stained with anti-CD3 and anti-CD4 and ran on the BD FACS Canto II (BD Biosciences, Franklin Lakes, New Jersey, USA). Samples with an average purity of ≥ 95% were considered for stimulation. CD4 + T cells were then stimulated with anti-CD3 (eBioscience Clone CD28.2) and anti-CD28 (eBioscience clone OKT3) at a 5 µg/ml concentration each and incubated for 48 h at 370 C in a CO2 incubator. After incubation, cells were washed and stained for CCR5 phenotyping monoclonal antibodies. Cells were stained with Zombie aqua and antibodies against CD3, CD4, and CCR5 (BD bioscience, San Jose, CA, USA) and were acquired on an eight-color FACS CANTO II (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo version 10.1 (San Carlos, CA, USA). The gating strategy used is shown in Additional Fig. 1 .
8
Expansion and Purification of CD3+ T Cells from AML Patients
9
Induction of Regulatory T Cells
10
Activation of T Cells with Anti-CD3/CD28
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We prepared stimulatory antibodies; Anti-CD3 (eBioscience Clone CD28.2) and anti-CD28 (eBioscience clone OKT3) at a concentration of 5µg/ml each. A clearly mapped out 96-well plate was used. The plate was coated by adding 100 µl of anti-CD3 at a concentration of 5ug/ml and incubated for 2 hours. After incubation, the plate was blotted and 100,000 cells in 90µl per sample were added. Using a pipette, 110 µl anti-CD28 was added to each well to make a total nal volume of 200µl at a concentration of 5ug/ml. For the negative control wells, 110µl of PBS was added to make volume of 200µl per well. The plate was incubated for a total of 48 hours at 37 0 C in a CO 2 incubator. After incubation, cells were washed with 200 µL staining buffer per well and then transferred to the 5 mm round bottomed polystyrene FACS tubes.
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