The TFH cell differentiation assay was performed with freshly isolated PBMCs from healthy donors (n = 16). Naïve CD4+ T cells were isolated by using the human Naïve CD4+ T Cell Isolation Kit II (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). A total of 500,000 naïve CD4+ T cells were cultured in complete medium (RPMI 1640 with 10% fetal calf serum, 1% penicillin/streptomycin solution and 1.5% 1M HEPES; all Thermo Fisher Scientific) in the presence of 12.5 μL/mL anti-CD3/CD28 (STEMCELL Technologies, Vancouver, Canada) in a flat-bottom 48 well plate. After 24 hours of culture, cytokines were added and cells were incubated for additional 2.5 days. A total of 5 ng/mL IL-12 + 5 ng/mL TGFβ with and without 100 ng/mL UST and 100 ng/mL UST alone were used.
Anti cd3 cd28
Manufactured by STEMCELL
Sourced in Canada
Anti-CD3/CD28 is a laboratory equipment product that activates T cells. It consists of antibodies that bind to the CD3 and CD28 receptors on the surface of T cells, providing essential signals for T cell activation and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Calf serum, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
T cell expansion medium, by STEMCELL (1 mentions)
α human igg1, by R&D Systems (1 mentions)
Anti pd 1 mab, by R&D Systems (1 mentions)
Anti tigit mab, by R&D Systems (1 mentions)
Penicillin, by Solarbio (1 mentions)
Streptomycin, by Solarbio (1 mentions)
4 protocols using anti cd3 cd28
1
Naïve CD4+ T Cell Differentiation
2
Effect of Ustekinumab on Activated PBMCs
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Two million PBMCs isolated from healthy donors were seeded in a sterile 24-well flat-bottom plate containing complete medium (RPMI 1640 with 10% fetal calf serum, 1% penicillin/streptomycin solution, and 1.5% 1M HEPES; all Thermo Fisher Scientific). Cells were activated with 25-μL/mL anti-CD3/CD28 (STEMCELL Technologies). Ustekinumab was added in 3 different concentrations (10 mg/L, 1 mg/L, or 0.1 mg/L), and cells were cultured for 7 days. Cells were analyzed by flow cytometry on days 0, 3, 5, and 7.
3
Isolation, Activation, and Expansion of CD8+ T Cells
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Human PBMCs were isolated from healthy donors by the density gradient centrifugation using Ficoll-Paque Plus from Sigma-Aldrich (#GE17-1440-02). Human CD8+ T cells were isolated from whole blood from healthy donors by the magnetic bead separation using the Direct Human CD8 Isolation Kit from Stemcell (#19663). Purified CD8+ T cells were activated and expanded with anti-CD3/CD28 (Stemcell, Canada, #10971) and cultured in RPMI 1640 containing 10% FBS, 100 µg/mL penicillin/streptomycin and recombinant human IL-2 (PeproTech). 72 h after activation, activated CD8+ T cells were treated with different conditioned medium (CM).
4
Regulation of CD8+ T Cells by CD155 and Checkpoint Inhibitors
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Mouse cervical cancer cell line U14 was obtained from the National Biomedical experimental cell resource bank (Beijing, China). U14 was cultured in DMEM supplemented with 10% fetal bovine serum (all from Gibco, Grand Island, NY, USA), 50 U/mL penicillin and 50 mg/mL streptomycin (all from Solarbio Science & Technology, Beijing, China). CD8 + T cells were puri ed from PBMC positive selection by using a kit (Milteny Biotec, Bergisch Gladbach, Germany). CD8 + T cells were stimulated with anti-CD3/CD28(Stemcell, Canada) in T cell expansion medium (Stemcell, Canada). Activated CD8 + T cells were treated with 5µg/mL CD155-Fc, 10µg/mLCD155-Fc. Activated CD8 + T were cocultured with tumor cells at a 10:1 ratio. Add 10µg/ml anti-PD-1 mAb or 5µg/ml anti-TIGIT mAb (R&D Systems), respectively. As isotype control, we used α-human IgG1 (R&D Systems). After 48 hours, CD8 + T cells were collected to determine cytokine production using the T cell function assay.
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