For coculture experiments, 2.5 × 105 pan T cells were cocultured with varying numbers of MDSCs in RPMI 1640 media supplemented with 1% l-glutamine, 1% penicillin/streptomycin, 10% heat-inactivated FBS, and CD3 and CD28 antibodies (T cell activation kit, miltenyi biotec) to activate mouse T cells. After 72 hours, cells were collected, and eFluor 670 dilutions were calculated by gating from live CD4+ or CD8+ T cells using flow cytometry.
T cell activation kit
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The T cell activation kit is a laboratory product designed to facilitate the activation and expansion of T cells, a type of immune cell. The kit provides the necessary components to stimulate T cell activation and proliferation in a controlled in vitro environment. The core function of the T cell activation kit is to enable researchers to study and manipulate T cell responses for various applications in immunology and cell biology research.
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Lab products found in correlation
Pan t cell isolation kit, by Miltenyi Biotec (2 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Cd4 microbeads, by Miltenyi Biotec (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Hepes, by Lonza (1 mentions)
Midimacs separator, by Miltenyi Biotec (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Ficoll, by Eurobio Scientific (1 mentions)
9 protocols using t cell activation kit
1
T Cell Proliferation Assay with MDSCs
2
Quantifying NFAT Activation in T Cells
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WT and Gal-3−/− T cells were isolated from healthy murine spleens using pan T cell isolation kit (Miltenyi). Isolated T cells were activated with a T cell activation kit (Miltenyi; CD3/CD28 antibodies conjugated to the beads). After two hours of activation, cells were stained for surface markers for 20 min, washed, and fixed in 2% formaldehyde in 1x PBS (40 min), the cells were then permeabilized in 5% normal mouse serum in 1x PBS +0.4% Triton X-100. Next, the cells were stained for intracellular NFAT for 20 min using a rabbit anti-human NFAT1 antibody. After washing once with NGS block (5% normal goat serum in 1X PBS), the cells were incubated with 2 μg/mL Alexa Fluor 647-conjugated donkey anti-rabbit IgG for 15 min. Before running samples on the ImageStream instrument (Amnis® ImageStream®XMark II), 10 μL of 0.5 μg/mL DAPI (nuclear stain) was added to each sample and incubated at room temperature for at least 5 min.
3
Assessing HIV-1 Infectivity in HeLa Cells
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HeLa P4R5 cells, a HeLa-CD4/LTR-lacZ indicator cell line expressing both CXCR4 and CCR5, were employed to assess viral infectivity (65 (link)) using a beta-galactosidase assay. 293T cells (ATCC) are human embryonic kidney cells used to produce lentiviral vectors and HIV-1 viruses; HeLa cells (ATCC) are derived from cervical cancer cells. CD4+ T cells were isolated from healthy donors and obtained via the Etablissement Français du Sang (EFS), Paris, France. Briefly, primary CD4+ T cells were purified from human peripheral blood by density gradient centrifugation (lymphocyte separation medium; PAA), followed by positive immunomagnetic selection with CD4 microbeads (Miltenyi). A day later, cells were activated by a T cell activation kit (Miltenyi) for 2 to 3 days at 37°C with interleukin-2 (IL-2)-containing medium (50 IU/ml); then cells were challenged with HIV-1 carrying the CA wild type or mutant. The percentage of p24-positive cells was obtained by cytofluorimetry acquisition and FlowJo analysis.
4
Co-culture of Gastric Cancer Spheroids and T Cells
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For the co-culture of GC spheroids and T cells, MKN74 (intestinal-type) and MKN45 (diffuse-type) GC spheroids were transferred to round-bottom 96-well plates, and Tregs/conventional T cells were added to the culture media at 1:1, 1:5, and 1:15 (GC cell:T cell) ratios. Co-cultures were maintained in RPMI 1640 with 25 mM HEPES (Lonza, Basel, Switzerland) supplemented with 10% FBS, 1% PS, 1% Sodium pyruvate (Sigma-Aldrich) and recombinant human IL-2 (10 ng/mL; PeproTech, London, UK). Treg cultures were further supplemented with anti-CD3/anti-CD28 MACSiBead (0.5 beads/T cell; T Cell Activation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany). As a control, MKN74/MKN45 single cultures (1:0; GC cell:T cell) were maintained in the same conditions as co-cultures. For all co-culture experiments (Figure 3 , Figure 4 and Figure 5 ), a minimal number of 6 independent T cells donors, collected and analyzed in at least 3 independent days are represented.
5
Cryopreserved MDSC-T Cell Co-culture
6
T Cell Proliferation Assay with MDSCs
7
Activated Mouse T Cell Cytokine Assay
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Mouse T cells were isolated from splenocytes of wild-type female BALB/c mice by Pan T cell isolation kit, a LS Column and a MidiMACS Separator (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany). The isolated T cells were then activated by using T cell Activation kit (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany). In brief, 1×106 T cells were incubated with anti-mouse CD3 and anti-mouse CD28 antibody-coated beads in the presence or absence of 5 μg/ml Fgl1 recombinant protein and cultured in 48-well plates at 37°C for 3 days. The culture supernatant was obtained at 72 h following stimulation, and the levels of IL-2 (Invitrogen, Calsbad, CA) or IFNγ cytokines (R&D systems, Minneapolis, MN) were measured using ELISA kits according to the manufacturer’s manual process.
8
Cryopreserved MDSC-T Cell Co-culture
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Cryopreserved MDSCs from an individual TB participant, with their corresponding adherent monocytes, as well as an aliquot of allogeneic T-cells isolated from isolated from a QFN positive donor, was removed from liquid nitrogen (LN) storage, thawed, washed and counted. These cells were cultured according the plate layout in Supplementary Fig. 6 . Briefly, combinations of 2×105 cells per well, were cultured in RPMI + 10% autologous human serum (AHS) + 1% L-glutamine (L-glut) with or without 1uM ATRA, under 2 T cell conditions including Unstimulated and non-specifically activated (anti-CD3CD28 beads, Miltenyi T-cell activation kit) T cells for 48 h at 37 °C with 5% CO2.
For trans-well co-cultures, 96 well 0.4 um Polycarbonate Membrane Corning Trans-well culture plate inserts (Becton Dickinson, New Jersey, USA) were used to separate MDSCs (top) and T-cells (bottom), following the plate layout inSupplementary Fig. 6 . Brefeldin-A was added to all wells, co-culture and trans-well cocultures, at 43 h to facilitate accumulation of proteins in the endoplasmic reticulum (ER) and prevent export of cytokine proteins from the cell. Culture supernatants were harvested at 48 h and stored at −80 °C for multiplex analysis of soluble cytokine production, and the cellular fraction cryopreserved in LN.
For trans-well co-cultures, 96 well 0.4 um Polycarbonate Membrane Corning Trans-well culture plate inserts (Becton Dickinson, New Jersey, USA) were used to separate MDSCs (top) and T-cells (bottom), following the plate layout in
9
Expansion and Stimulation of CSF-infiltrating CD4+ T Cells
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PBMCs were freshly isolated using Ficoll (Eurobio, Germany) density-gradient centrifugation. CSF-infiltrating CD4+ T cells were expanded with phytohaemagglutinin in a single round of stimulation using a previously reported protocol aimed to reduce T cell receptor repertoire bias.18 (link) 6 × 104 phytohaemagglutinin-expanded CSF-infiltrating CD4+ T cells were seeded in quadruplicate with 2 × 105 irradiated autologous PBMCs as antigen-presenting cells and stimulated with peptides (eTable 2, links.lww.com/NXI/A590 ) at a final concentration of 10 μM and with a T Cell Activation Kit (anti-CD3, anti-CD28, and anti-CD2 beads) (Miltenyi Biotec) as positive control.
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