CD8+ T cells were purified from the spleen using magnetic separation (Miltenyi Biotec), cultured with 1 μg/ml plate-bound anti-CD3 (eBioscience) and 2 μg/ml soluble anti-CD28 (eBioscience), in a 96-well plate with 200 μl of RPMI supplemented with 10% FCS, 2 mM L-glutamine, 5 x 10-5M 2-mercaptoethanol, 1 mM sodium pyruvate, and 0.1 mM non-essential amino acids. 5 x 104 CD8+ T cells were cultured in each well. Cell blast formation occurred 24 h after cell culture. Cell numbers became 6.5 x 105 after 72-hour-culture and dead cells were observed below 10% under microscope by Trypan blue staining. For the metformin-treated group, cells were treated with metformin at 0, 1, 10, 100, 1000, or 5000 μM. Cells were collected at 48, 96, 120h for extracellular flux analysis or flow cytometry.
Plate bound anti cd3
Manufactured by Thermo Fisher Scientific
Plate-bound anti-CD3 is a lab equipment product used to activate T cells. It binds to the CD3 receptor on the surface of T cells, which triggers their activation and proliferation. The product is designed for in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Soluble anti cd28, by Thermo Fisher Scientific (4 mentions)
Mouse cd4 cd25 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Mouse cd4 cd62lhi t cell isolation kit, by Miltenyi Biotec (2 mentions)
Magnetic separation, by Miltenyi Biotec (1 mentions)
Human cd4 microbeads, by Miltenyi Biotec (1 mentions)
Soluble anti cd28, by BD (1 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Gw788388, by Merck Group (1 mentions)
X vivo 15 media, by Lonza (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
5 protocols using plate bound anti cd3
1
Metformin Modulates CD8+ T Cells
2
Isolation and Activation of Mouse CD4+ T Cells
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Naïve CD4+ T cells were isolated from mouse spleens using a mouse CD4+CD62Lhi T Cell Isolation Kit according to the manufacturer's instructions (Miltenyi Biotec). Splenic CD4+CD25+ Tregs were isolated using a mouse CD4+CD25+ T cell isolation kit according to the manufacturer's instructions (Miltenyi Biotec). Purified cells (approximately 0.5 × 106 cells/mL) were cultured at 37°C in RPMI 1640 containing 10% fetal calf serum, penicillin/streptomycin, and 50 μmol/L 2‐mercaptoethanol with 1 μg/mL plate‐bound anti‐CD3 (eBioscience) and 1 μg/mL soluble anti‐CD28 (eBioscience) for 3 days, as indicated in each experiment. For Th17 differentiation, 2 ng/mL TGF‐β1 and 50 ng/mL IL‐6 were added.
3
Trans-endothelial Migration Assay
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The transwell assay in this study was slightly modified from previously described methods (30 –32 ). First, blood CD4+ T cells from healthy donors were isolated with human CD4 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany, 130-045-101) according to the manufacturer’s instructions. Isolated CD4+ T cells were cultured in the above-mentioned medium and incubated in a 24-well plate with 5×105 cells per well. CD4+ T cells were stimulated with plate-bound anti-CD3 (eBioscience, Carlsbad, CA, 16-0037, 0.25ug/ml) and soluble anti-CD28 (eBioscience, Carlsbad, CA, 16-0289, 1ug/ml) for 48 hours. Next, activated CD4+ T cells were harvested and placed on the upper chamber of a 24-well transwell plate with 5µm pores (Corning, Kennebunk, MA, 3421) for 24 hours. To investigate whether ALCAM induces trans-endothelial migration of T cells, rhALCAM (3ug/ml) was added to the lower chamber, with vehicle (1× PBS) as control. Then cells in lower chamber were counted with hemacytometers.
4
Modulating T cell differentiation
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Primary dermal fibroblasts from ERBB2IPmut, STAT3mut, or healthy donors were grown in MEM supplemented with 20% fetal bovine serum and penicillin-streptomycin-glutamine (Thermo Fisher Scientific). 293T cells were cultured in IMDM with 10% FBS and penicillin-streptomycin-glutamine. Transfection of 293T cells with plasmids or siRNAs was accomplished with Nucleofector V (Lonza). Isolated naive primary CD4+ T cells were cultured in X-VIVO 15 media (Lonza). Plate-bound anti-CD3 (5 µg/ml unless otherwise indicated; eBioscience), soluble anti-CD28 (1 µg/ml; BD), and IL-2 (20 U/ml unless otherwise indicated; PeproTech) were used in nonskewing and iT reg cell conditions; TGF-β1 (R&D Systems) was used in iT reg cell cultures as indicated. When specified, a neutralizing anti-(α)TGFβ1 antibody (20 µg/ml; R&D Systems), a small molecule ALK5 inhibitor (TGFβR1i) GW788388, or a selective SMAD3 inhibitor (SMAD3i) SIS3 (Sigma-Aldrich) were added at 1 and 5 µM to nonskewing cultures of naive CD4 cells. To test specificity and inhibitory activity on TGF-β pathway activation, these inhibitors were added to the 293T SMAD-reporter line for 12 h before stimulation with TGF-β at the indicated concentrations.
5
Isolation and Culture of Murine and Human Naive T Cells and Tregs
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The mouse CD4+CD62Lhi T Cell Isolation Kit was used to isolate naïve CD4+ T cells from mouse spleens according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Splenic CD4+CD25+ Tregs were isolated using the mouse CD4+CD25+ T Cell Isolation Kit according to the manufacturer’s instructions (Miltenyi Biotec). Purified cells (0.5 × 106 cells/ml) were cultured at 37°C in RPMI-1640 containing 10% fetal calf serum, penicillin/streptomycin, and 50 μM 2-mercaptoethanol with 1 μg/ml plate-bound anti-CD3 (eBioscience) and 1 μg/ml soluble anti-CD28 (eBioscience) for 18 h to 3 days, as indicated in each experiment. For Treg differentiation, 2 ng/ml recombinant human TGF-β1 (Peprotech, Rocky Hill, NJ) was added to the cultures. Purified human naïve CD4+ T cells (0.4 × 106 cells/ml) were cultured at 37°C in X-VIVO 15 using Dynabeads™ Human T-activator CD3/CD28 (Thermo Fisher Scientific) for 3 days. For human Treg differentiation, 2 ng/ml recombinant human TGF-β1 (Peprotech) was added to the cultures.
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