Cryopreserved human peripheral blood mononuclear cells from three healthy donors were thawed and activated with anti-CD3 (clone OKT3) and anti-CD28 (clone 28.2) antibodies (Biolegend) and cultured with IL-7 (10 ng / mL) and IL-15 (5 ng / mL) (R&D System). PBMCS were transduced on day three with the RVSFG.CD19.CD28.4-1BBzeta retroviral vector (kindly provided by M Brenner, Baylor College of Medicine, Houston, Texas) using spinoculation on retronectin-coated (3.5 µg / mL, Takara) 24-well plates. CAR-T cells were maintained in cytokine-supplemented media (ImmunoCult™-XF T Cell Expansion Medium, Stemcell Technologies) and used for experiments 10 days after transduction.
Anti cd28 clone 28
Manufactured by BioLegend
Sourced in United States
Anti-CD28 (clone 28.2) is a mouse monoclonal antibody that recognizes the CD28 antigen. CD28 is a costimulatory molecule expressed on T cells that plays a crucial role in T cell activation and function.
Automatically generated - may contain errors
Lab products found in correlation
Alpn 202, by BioLegend (2 mentions)
Retronectin coated, by Takara Bio (1 mentions)
Anti cd3 clone okt3, by BioLegend (1 mentions)
Immunocult xf t cell expansion medium, by STEMCELL (1 mentions)
Il 15, by R&D Systems (1 mentions)
Interleukin 7 (il 7), by R&D Systems (1 mentions)
Anti cd3 clone okt3, by BioXCell (1 mentions)
Clone okt3, by BioLegend (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Cd4 bv421, by BioLegend (1 mentions)
Cd8 percp, by BioLegend (1 mentions)
Golgistop, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Anti cd3, by BioLegend (1 mentions)
Legend max human il 2 elisa kit, by BioLegend (1 mentions)
Anti ctla4, by BioLegend (1 mentions)
Alexa fluor antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
6 protocols using anti cd28 clone 28
1
Activation and Transduction of CAR-T Cells
2
T cell Activation in SLE Patients
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Isolated T cells from SLE patients and healthy controls were stimulated with pre-coated 1 μg ml−1 anti-CD3 (clone OKT3, BioXcell) and 1 μg ml−1 anti-CD28 (clone 28.2, BioLegend) for 4 days.
3
T Cell Activation Methods
4
T Cell Degranulation Assay by CD107a
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To measure degranulation via CD107a staining, PBMCs were cultured in supplemented RPMI medium containing IL-2 (50 IU/mL, Peprotech, NJ, USA) and anti-CD3 (OKT3, Ortho Pharmaceutical Corporation, NJ, USA) and anti-CD28 (clone 28.2, BioLegend)-coated g'a'm-Dynabeads (Thermo Fisher Scientific) were added in a 1:1 ratio. T cells were cultured for 48 h in 24-well plates at a concentration of 1 Â 10 6 cells/mL. Subsequently, 1 Â 10 5 PBMCs were cocultured with K562-S, K562-S-PD-L1, and K562-S HVEM (2 Â 10 4 ) for 5 h (total volume, 100 mL/well). An anti-CD107a antibody (H4.A3, FITC labeled, BioLegend) was added together with Golgistop and Golgiplug (BD Bioscience) at the onset of coculture. Subsequently, cells were stained for expression of CD4 and CD8 (CD4-BV421, CD8-PerCP, both BioLegend) and analyzed by flow cytometry. Sample acquisition was performed using constant volumes, flow rates, and acquisition time for all samples. Luminexbased cytokine analyses were performed on a parallel coculture after 24-48 h as described above.
5
Modulation of T-cell Activation by ALPN-202
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K562 cells stably expressing membrane-anchored anti-CD3 single chain Fv (clone OKT3) and/or PD-L1 were plated at 12,500 cells/well in 96-well plate. 1 × 105 primary T cells (Bloodworks Northwest) and serial dilutions of each test article were added to each well. In some cases, titrations of ALPN-202 or monomeric CD80 vIgD were combined with 100 nM blocking anti-PD-L1 or anti-CD28. Plates were incubated 24 h at 37 °C. Secreted IL-2 was measured using a LEGEND MAX™ Human IL-2 ELISA kit (BioLegend).
In separate experiments, 1 × 105 primary T cells were co-cultured with 2 × 104 K562/PD-L1 cells and incubated with a 100 nM–0.02 nM ALPN-202, agonist anti-CD28 (clone 28.2, BioLegend), Fc control, anti-PD-L1 (atezolizumab), anti-CTLA-4 (ipilimumab), or a combination of anti-PD-L1 and anti-CTLA-4. Submaximal TCR stimulation was provided by 12.5, 2.5, 0.5, or 0 nM anti-CD3 (clone OKT3, BioLegend). After 16 h supernatants were collected and analyzed for secreted IL-2. Cells were then treated with brefeldin A/monensin for 6 h and characterized by flow cytometry for intracellular IL-2 and CD25 upregulation. Four donors were tested in two separate experiments with all samples run in triplicate.
In separate experiments, 1 × 105 primary T cells were co-cultured with 2 × 104 K562/PD-L1 cells and incubated with a 100 nM–0.02 nM ALPN-202, agonist anti-CD28 (clone 28.2, BioLegend), Fc control, anti-PD-L1 (atezolizumab), anti-CTLA-4 (ipilimumab), or a combination of anti-PD-L1 and anti-CTLA-4. Submaximal TCR stimulation was provided by 12.5, 2.5, 0.5, or 0 nM anti-CD3 (clone OKT3, BioLegend). After 16 h supernatants were collected and analyzed for secreted IL-2. Cells were then treated with brefeldin A/monensin for 6 h and characterized by flow cytometry for intracellular IL-2 and CD25 upregulation. Four donors were tested in two separate experiments with all samples run in triplicate.
6
Blockade of Immune Checkpoint Interactions
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CD80-Fc and PD-1-Fc proteins (R&D Systems) were conjugated with Alexa Fluor® 647 (Molecular Probes® Alexa Fluor® Antibody Labeling Kit, Thermo Fisher Scientific) according to manufacturer’s instructions. To measure blockade of PD-1 - PD-L1, K562/PD-L1 cells were plated in a 96-well plate and incubated with serial dilutions of ALPN-202, anti-PD-L1 (atezolizumab), or Fc control. Cells were washed and incubated with PD-1-Alexa Fluor 647. Bound PD-1 was detected using a Becton Dickinson LSRII flow cytometer and data analyzed using FlowJo v7 software. To measure blockade of CD28 - CD80 and CTLA-4 - CD80, CHO cells stably expressing CD28 or CTLA-4 were plated in 96-well plates and incubated with serial dilutions of ALPN-202, anti-CD28 (clone 28.2, BioLegend), anti-CTLA-4 (ipilimumab) or Fc control. Cells were washed and incubated with CD80-Alexa Fluor 647. Bound CD80 was detected using a Becton Dickinson LSR II flow cytometer and data analyzed using FlowJo v7 software.
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