The human T cells were isolated from the three healthy volunteers using a human T cell isolation kit (Miltenyi Biotec) according to the manufacturer’s protocol. T cells were cultured in Human ImmunoCult-XFT Cell Expansion medium (Stem Cell) with penicillin (100 u/mL) and streptomycin (100 mg/mL) at 37°C with 5% CO2. T cells were prestimulated with IFN-γ (Peprotech) for 48 hours before co-culture with MMAC-SF cells. After 24 hours of co-culture, PD1+ T cells apoptosis was determined by flow cytometry.
Human immunocult xf t cell expansion medium
Manufactured by STEMCELL
Human ImmunoCult-XF T Cell Expansion medium is a serum-free, xeno-free culture medium designed for the expansion of human T cells in vitro. The medium provides the necessary components to support the growth and proliferation of human T cells without the use of animal-derived components.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Human pan t cell isolation kit, by Miltenyi Biotec (2 mentions)
Anti cd3, by Thermo Fisher Scientific (2 mentions)
Anti cd28, by Thermo Fisher Scientific (2 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Human t cell isolation kit, by Miltenyi Biotec (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Easysep human cd8 t cell isolation kit, by STEMCELL (1 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Recombinant dkk1, by Thermo Fisher Scientific (1 mentions)
Pembrolizumab, by Merck Group (1 mentions)
4 protocols using human immunocult xf t cell expansion medium
1
Examining PD1+ T Cell Apoptosis
2
Expansion of Tumor-Infiltrating T Cells
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Peripheral blood mononuclear cell (PBMC)-derived T cells from 4 MSI-H CRC patients were isolated, and TILs from surgical-resected tissues were obtained after PD-1 blockade, using a human pan-T cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec). Human T cells were cultured in Human ImmunoCult-XF T Cell Expansion medium (Stem Cell) with penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37 °C with 5% CO2. Cells were prestimulated with IL-2 (200 U/ml, Peprotech), anti-CD3 (5 µg/mL, Peprotech) and anti-CD28 (5 µg/mL, Peprotech) for 48 h in the presence of paired CRC organoid cells at a 20:1 ratio.
3
Isolation and Activation of Human CD8+ T Cells for Tumor Cell Co-culture
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According to the manufacturer’s instructions, human T cells were obtained from 3 healthy volunteers’ PBMCs (07851, Stemcell). And we used EasySepTM Human CD8+ T Cell Isolation Kit (17953, Stemcell) to isolate the CD8+ T cells from PBMCs. CD8+ T cells were cultured in the Human ImmunoCult-XF T Cell Expansion medium (10981, Stemcell) with 1% Penicillin-Streptomycin (15140122, Gibco). The CD8+ T cells were cultured in the medium at 1 × 106/mL. To activate T cells, 2 μg/mL of anti-CD3 (02121-25-500, Peprotech), 1 μg/mL of anti-CD28 (10311-25-500, Peprotech), and 200 U/mL of IL-2 (200-02-10, Peprotech) were added to the cell suspension for 3 days. HCT116 cell line was purchased from the Chinese Academy of Sciences (Shanghai, China). DMEM (11995065, Gibco) with 10% fetal bovine serum (SA101, Cellmax) and 1% Penicillin-Streptomycin (15140122, Gibco) were used to culture tumor cells. After being prepared as described above, CD8+ T cells were directly added to tumor cells at a 5:1 ratio to establish the CD8+ T cell-tumor cell co-culture system.
4
Durable Immune Responses in dMMR CRC
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PBMCs from two patients with dMMR CRC (dMMR1 and dMMR2) and healthy volunteers were isolated, and then T cells and CD8+ T cells were isolated from the PBMCs using a human pan-T cell isolation kit and CD8+ T-cell isolation kit, each according to the manufacturer’s instructions (Miltenyi Biotec). Human T cells were cultured in Human ImmunoCult-XF T Cell Expansion medium (Stem Cell) with penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37°C with 5% CO2. Cells were prestimulated with IL-2 (200 U/mL, Peprotech), anti-CD3 (Peprotech) and anti-CD28 (Peprotech) for 48 hours before treatment with recombinant DKK1 (Peprotech) and DKK1 neutralizing antibody (Sinobio) or IgG. Before the experiment, T cells from patients with dMMR were also prestimulated in the presence of paired CRC organoid cells for 48 hours. CD8+ T cells were cultured in the same culture medium and conditions as the T cells. For PD-1 neutralization, T cells were treated with 1 µg/mL pembrolizumab (Keytruda, Merck & Co.) for 24 hours.
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