CD4+ T cells were isolated from mouse spleen using CD4 MicroBeads (Miltenyi) according to the manufacturer’s protocol. To measure apoptosis, T cells were stimulated with anti-CD3/anti-CD28 antibodies (eBioscience) for 3.5 d, stained with Annexin V (BD Phar-Mingen), and the apoptotic cells were analyzed by flow cytometry (BD LSR II). For the proliferation assay, splenic CD4+ T cells were stained with 5 μM CellTrace Violet reagent (Thermo Fisher Scientific) prior to stimulation with anti-CD3/anti-CD28 and successive generations of live cells were analyzed by flow cytometry. Proliferation was quantified using cell populations that underwent more than 5 divisions normalized to total live cells. Senescence in MEFs was quantified by staining for b-galactosidase activity as previously described (Senescence β-Galactosidase Staining Kit, #9860, Cell Signaling Technology) (Park et al., 2018 (link)). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in MEFs were measured by Seahorse XF 24 Analyzer (Agilent) as previously described (Kang et al., 2014 (link)).
Anti cd3 anti cd28 antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Anti-CD3/anti-CD28 antibodies are immunological reagents used to activate T cells in vitro. They bind to the CD3 and CD28 surface receptors on T cells, providing the necessary signals to stimulate T cell proliferation and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Senescence β galactosidase staining kit, by Cell Signaling Technology (2 mentions)
Celltrace violet reagent, by Thermo Fisher Scientific (2 mentions)
Cd4 microbeads, by Miltenyi Biotec (2 mentions)
Lsr 2, by BD (2 mentions)
Annexin 5, by BD (2 mentions)
Seahorse xf24 analyzer, by Agilent Technologies (2 mentions)
Recombinant il 2, by Thermo Fisher Scientific (2 mentions)
Recombinant tgf β, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (2 mentions)
T cell isolation kit, by Miltenyi Biotec (1 mentions)
Lysis fix buffer, by BD (1 mentions)
Huh7 cell line, by RIKEN Cell Bank (1 mentions)
Aim 5, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Plc prf 5, by American Type Culture Collection (1 mentions)
Sk hep1, by American Type Culture Collection (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
10 protocols using anti cd3 anti cd28 antibodies
1
Measuring T Cell Apoptosis and Proliferation
2
SAA-Induced CD4+ T Cell Response
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The spleen was ground completely in RPMI-1640 media plus 1% antibiotic and 10% of fetal bovine serum, followed by expelling several times with a 19-G needle and filtering with a mesh nylon screen to collect single cells. Then, CD4+ T cells were isolated from mononuclear cells by using a T-cell isolation kit (Miltenyi Biotec). These cells (2 × 105) were treated with or without 50 ng/mL mouse recombinant SAA/SAA1 (R&D Systems, Minneapolis, MN, USA) for 48 hours in the presence of anti-CD3/anti-CD28 antibodies from eBioScience (San Diego, CA, USA). In addition, CD4+ T cells were stimulated with 50 ng/mL SAA1 for 24 hours in the presence of anti-CD3/anti-CD28 antibodies, followed by flow cytometry.
3
Induction of Human Regulatory T Cells
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CD4+CD25− T cells were activated in the presence of plate-bound anti-CD3/anti-CD28 antibodies (eBiosciences, UK) with a ratio of 1:2::anti-CD3:anti-CD28 (1.0 μg/ml anti-CD3: 2.0 μg/ml anti-CD28) for iTregs. Briefly, naïve T cells were differentiated into iTregs using 5.0 ng/ml recombinant-TGF-β, 10.0 ng/ml recombinant-IL-2 (eBiosciences, Germany), cultured for 3–4 days in RPMI1640 medium (Invitrogen) supplemented with FBS, Penicillin/Steptomycin, L-glutamine, β-Mercapto ethanol55 (link). Cells were harvested at day 3–4 and used for intracellular staining using flow cytometry, q-RT-PCR and immune-blotting experiments.
4
Isolation and Activation of PBMCs
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PBMCs were isolated by density gradient centrifugation (1 × 106 cells/ml RPMI, 10% fetal calf serum (FCS) + 1% penicillin/streptomycin), stimulated with anti-CD3/anti-CD28 antibodies (eBioscience, San Diego, CA, USA) at 37 °C, 5% CO2 for 24 h. The next day, cells were harvested, washed (500× g, 5 min) an incubated with lysis/fix buffer (BD Biosciences) for 12 min at 37 °C. After washing (500× g, 5 min) cells were stained as described above. An unstimulated panel was used as negative control.
5
Measuring T Cell Apoptosis and Proliferation
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CD4+ T cells were isolated from mouse spleen using CD4 MicroBeads (Miltenyi) according to the manufacturer’s protocol. To measure apoptosis, T cells were stimulated with anti-CD3/anti-CD28 antibodies (eBioscience) for 3.5 d, stained with Annexin V (BD Phar-Mingen), and the apoptotic cells were analyzed by flow cytometry (BD LSR II). For the proliferation assay, splenic CD4+ T cells were stained with 5 μM CellTrace Violet reagent (Thermo Fisher Scientific) prior to stimulation with anti-CD3/anti-CD28 and successive generations of live cells were analyzed by flow cytometry. Proliferation was quantified using cell populations that underwent more than 5 divisions normalized to total live cells. Senescence in MEFs was quantified by staining for b-galactosidase activity as previously described (Senescence β-Galactosidase Staining Kit, #9860, Cell Signaling Technology) (Park et al., 2018 (link)). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in MEFs were measured by Seahorse XF 24 Analyzer (Agilent) as previously described (Kang et al., 2014 (link)).
6
Differentiation of Naive T Cells into Induced Regulatory T Cells
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To differentiate naïve T cells into iTreg, plates were pre-treated with a ratio of 1:2:anti-CD3:anti-CD28 (1.0 µg/ml anti-CD3: 2.0 µg/ml anti-CD28) [11] . CD4 + CD25 -T cells were then activated after incubated 3-4 days in RPMI1640 medium (Invitrogen) supplemented with FBS, Penicillin/Steptomycin, L-glutamine, β-Mercaptoethanol [32] on the plate bound with anti-CD3/anti-CD28 antibodies (eBiosciences, UK), and 5.0 ng/ml recombinant-TGF-β, 10.0 ng/ml recombinant-IL-2 (eBiosciences, Germany) were added during the incubation. Ceramide C6 (Sigma, Germany) was used (1-10µM) together with TGF-β and IL-2 for iTreg differentiation. Cells were harvested at day 3-4 and used for flow cytometry, q-RT-PCR and immune-blotting experiments.
7
Establishment of GPC3-expressing Liver Cancer and NK Cell Cultures
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Huh7 cell line was obtained from the RIKEN Cell Bank. 293T, NK92 and other HCC cell lines (PLC/PRF/5, and SK-Hep-1) were purchased from the American Type Culture Collection (ATCC). Human NK92 cells were maintained in alpha minimum essential medium (GIBCO, USA) as described previously (39 (link)). SK-Hep-1 cells were lentivirally transduced to stably express human GPC3 using Pwpt-GPC3 lentiviral vectors (designated as “SK-Hep-1-GPC3”). 293T and HCC cells were cultivated in DMEM medium (GIBCO, USA) and 10% fetal bovine serum (FBS, GIBCO, USA).
Human peripheral blood mononuclear cells (PBMCs) were derived from healthy donors. Primary human T cells were isolated from PBMCs by density gradient centrifugation. Then T cells were stimulated with tosyl-activated paramagnetic beads that were immobilized with anti-CD3/anti-CD28 antibodies (Invitrogen) for 24 h and the cell: bead ratio was 1:1. T cells were cultured in human T cell medium AIM-V(GIBCO, USA) with 2% human AB serum and 300 IU/mL recombinant human IL2.
Human peripheral blood mononuclear cells (PBMCs) were derived from healthy donors. Primary human T cells were isolated from PBMCs by density gradient centrifugation. Then T cells were stimulated with tosyl-activated paramagnetic beads that were immobilized with anti-CD3/anti-CD28 antibodies (Invitrogen) for 24 h and the cell: bead ratio was 1:1. T cells were cultured in human T cell medium AIM-V(GIBCO, USA) with 2% human AB serum and 300 IU/mL recombinant human IL2.
8
Murine Immune Cell Isolation and Activation
9
Murine Immune Cell Isolation and Activation
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All data points referring to murine samples represent distinct mice. Murine splenocytes were extracted from spleens by mechanical dissociation and red blood cells were lysed with ACK lysis buffer (Gibco, A10492) for 5 min at room temperature. Naive CD4+ T cells were sorted on a BD FACSAria II or Melody cell sorter. The purity of sorted murine naive CD4+ and naive CD8+ T cells was routinely verified by flow cytometry (see Figure S4A ). For stimulation experiments, cells were activated with plate-bound anti-CD3/anti-CD28 antibodies (both 2.5 μg/mL, Invitrogen, #16-0033-85 and #16-0281-85) for 4 days. For adoptive transfer experiments, total mouse T cells were first isolated from splenocytes by the Pan T cell isolation kit II (Miltenyi Biotec, #130-095-130). Then, naive CD4+ and naive CD8+ CD44− T cells were sorted on a BD FACSAria II or Melody cell sorter. Murine thymocytes were extracted from thymi by mechanical dissociation and red blood cells were lysed with ACK lysis buffer (Gibco, A10492) for 1 min at room temperature.
10
CD3+ T Cell Proliferation Assay
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To evaluate CD3+ T cells’ proliferation, BrdU (Bromodeoxyuridine) was used, following the manufacturer’s instructions. T cells were co-cultured with MDSCs at the following ratios: 0:1, 1:1, and 1:2 in RPMI-1640 medium in transwell inserts. Anti-CD3/anti-CD28 antibodies (at bead/cell ratio of 1:1; Human T Cell Activator CD3/CD28 Dynabeads Invitrogen, Carlsbad, CA) was also supplied with 500 IU/ml of IL-2 (R&D Systems, Minneapolis, MN) and then applied to stimulate the CD3+ T cells. The levels of BrdU incorporation were calculated in T cells with a spectrophotometer at 450 nm.
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