The suppressive function of monocytic MDSC was evaluated by their ability to suppress the proliferation of autologous pan T cells. Untouched autologous T cells were isolated using a Rhesus Pan T cell isolation kit (Miltenyi Biotec), according to the manufacturer’s instructions. The T cells were labelled with Violet Proliferation Dye 450 (VPD450) (BD Pharmingen) according to the manufacturer’s instructions. VPD450-labelled T cells (1 × 105) were cultured in RPMI-1640 supplemented with 5% human AB serum (Gemini Bioproducts), penicillin/streptomycin (LONZA) and L-glutamine (CellGro) with (1 × 105) MDSC (1:1) or (5 × 104) MDSC (1:2) in 96-well, U-bottom plates. Microbead particles loaded with anti-monkey CD2, CD3 and CD28 Abs (Miltenyi Biotec T cell expansion kit) were used for T cell stimulation and added at 7.5 microbeads per T cell. The cultures were incubated for 4 days at 37°C in 5% CO2. T cell proliferation was determined by VPD450 dilution flow cytometric analysis. At the end of the culture period, T cells were harvested and the percentage and phenotype of proliferating T cells determined by FACS staining for naïve T cells (Tn), Tmem and Treg.
T cell expansion kit
Manufactured by Miltenyi Biotec
The T cell expansion kit is a laboratory product designed to facilitate the expansion of T cells in vitro. It provides the necessary components to support the growth and proliferation of T cells under controlled conditions.
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3 protocols using t cell expansion kit
1
Monocytic MDSC Suppression of T Cell Proliferation
2
Isolation and Activation of CD4+ T Cells for mTORC1 Targeting
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CD4+ T cells were isolated from PBMCs using a magnetic bead separation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described in our studies.33 , 35 Briefly, lineage‐specific biotin‐conjugated antibodies and anti‐biotin MicroBeads were used to remove non‐CD4+ T cells, and CD25+ PE‐labelled cells were magnetically removed from purified CD4+ fractions, leaving unlabeled CD4+CD25− cells for stimulation experiments. The CD4+ T cells were activated for 72 h in RPMI‐1640 medium supplemented with 10% FBS and a T Cell Expansion Kit (Miltenyi Biotec), containing biotinylated antibodies against human CD2, CD3 and CD28. For pathogen stimulation experiments, cells were stimulated with phytohemagglutinin (PHA, 5 μg mL−1, Sigma‐Aldrich, Missouri, USA) for 72 h, followed by a further sorting analysis. To target mTORC1, commercial non‐targeted siRNA, PTEN siRNA and PS6K siRNA were used following mitogenic stimulation according to the manufacturer's instructions (#6568, #6251 and #6566, Cell Signalling, Danvers, USA).
3
T-Cell Stimulation and Signaling Protocol
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T-cells were cultured in RPMI 1640 medium supplemented with antibiotics, 3% (for 18 h stimulation experiments) or 10% heat-inactivated FCS + 0,00035% 2-mercapto-ethanol for longer cultivation.
TCR stimulation with soluble cross-linking Abs was accomplished by addition of 1.7 µg/mL CD3ε and/or CD28 mAb, each. For higher-order cross-linking in solution biotinylated CD3ε and/or biotinylated CD28 mAb was used at the same concentration followed by addition of 5 µg/mL streptavidin. CD3ε and CD28 Abs were mixed before administration for combined stimulations. Stimulation with CD3ε and CD28 mAb coated beads was carried out as described in the manufacturer's manual to the Miltenyi T-cell expansion Kit. For the surface immobilised stimulation cell culture plates were coated with 5 µg/ml CD3ε mAb diluted in PBS 2 h, at 37°C and 5% CO2, then washed directly before seeding the cells in the presence of 1.7 µg/ml soluble CD28 mAb. T-cells from Tg(Nr4a1-EGFP/cre) mice were additionally stimulated with 10 µg LPS or 10 µg CpG, respectively. For the analysis of T-cell receptor signalling (see below) T-cells were stimulated with 1.5 µg/ml CD3ε and/or CD28 mAb. The same amounts were used for the biotinylated antibodies and, additionally 7.5 µg/ml streptavidin.
TCR stimulation with soluble cross-linking Abs was accomplished by addition of 1.7 µg/mL CD3ε and/or CD28 mAb, each. For higher-order cross-linking in solution biotinylated CD3ε and/or biotinylated CD28 mAb was used at the same concentration followed by addition of 5 µg/mL streptavidin. CD3ε and CD28 Abs were mixed before administration for combined stimulations. Stimulation with CD3ε and CD28 mAb coated beads was carried out as described in the manufacturer's manual to the Miltenyi T-cell expansion Kit. For the surface immobilised stimulation cell culture plates were coated with 5 µg/ml CD3ε mAb diluted in PBS 2 h, at 37°C and 5% CO2, then washed directly before seeding the cells in the presence of 1.7 µg/ml soluble CD28 mAb. T-cells from Tg(Nr4a1-EGFP/cre) mice were additionally stimulated with 10 µg LPS or 10 µg CpG, respectively. For the analysis of T-cell receptor signalling (see below) T-cells were stimulated with 1.5 µg/ml CD3ε and/or CD28 mAb. The same amounts were used for the biotinylated antibodies and, additionally 7.5 µg/ml streptavidin.
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