CD4+ T cells were seeded at 2.5×105 cells/well on 24-well plates (Starlab, Milton Keynes, UK) and cultured for 48 hours in the presence of 25% sera derived from patients or HCs (n=15 per group). The effect of IL-35 present in the sera (n=12) on driving an HLA-G-positive phenotype was tested through sera pretreatment with 0.5 µg/mL anti-IL-35 neutralising antibody (α-IL-35) (Bio-Techne, Abingdon, UK) prior to culture isolated CD4+ T cells for 45 min at room temperature. Similarly, controls were carried out in the presence or absence of anti-IL-10 neutralising antibody (α-IL-10, at 1 µg/mL) (Bio-Techne). The phenotype of the cells following sera conditioning was screened using flow cytometry.
24 well plates
Manufactured by Starlab
Sourced in United Kingdom
The 24-well plates are a type of laboratory equipment used for various cell culture and biochemical experiments. They are a multi-well cell culture plate with 24 individual wells, each capable of holding a small volume of liquid. These plates are commonly used for experiments that require parallel testing or screening of multiple samples or conditions.
Automatically generated - may contain errors
Lab products found in correlation
α il 35, by Bio-Techne (1 mentions)
Alexa 555 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Observer z1, by Zeiss (1 mentions)
Trypletm express, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated annexin 5, by Abcam (1 mentions)
Guava easycyte 8ht flow cytometer, by Merck Group (1 mentions)
3 protocols using 24 well plates
1
Modulation of CD4+ T Cell Phenotype by Sera
2
Immunocytochemistry of Transcription Factor
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MV3 cells were seeded on coverslips in 24-well plates (Starlab GmbH) overnight. After washing with DPBS, cells were fixed with 4% paraformaldehyde for 10 min at RT. Prior to staining, cells were permeabilized with 0.1% saponin for 15 min. Afterwards, cells were stained with TF antibody (rabbit anti-mouse and human, IgG 1:100, GeneTex GTX01033) for 1 h at RT. After washing with DPBS three times, cells were further stained with the secondary antibody: Alexa 555-conjugated goat anti-rabbit (IgG, Thermo Fisher Scientific, 1:500) for 45 min at RT. Nuclei were stained with DAPI for 20 min at RT. Finally, samples were imaged with a fluorescence microscopy (Observer z.1, Zeiss).
3
Quantifying Apoptosis, Necrosis, and Viability
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For quantifying apoptotic, necrotic and viable cells by the flow‐cytometric annexin V/PI assay, SW480, HCT116, HCT116p53‐ko, HCT116bax‐ko cells were seeded in densities of 7×104 cells/well into 24‐well plates (Starlab) and 24 h later exposed to compounds 2 and 3 at concentrations of 0.4 μm , 2 μm , 10 μm and 50 μm for 24 h. After treatment, the drug‐containing media were collected, cells were washed once with 37 °C warm PBS, and trypsinized for 5 min with TrypLETM Express (Gibco). Following trypsinisation, the cell suspensions were added to the pre‐collected media, and cells were pelleted by centrifugation (300 g, 3 min). The supernatants were discarded and cell pellets were resuspended with FITC‐conjugated annexin V (0.4 μg mL−1; BioVision, USA) in binding buffer (10 mm HEPES/NaOH pH 7.4, 140 mm NaCl, 2.5 mm CaCl2) and incubated at 37 °C for 15 min. Cells were subsequently stained with PI (propidium iodide, 1.6 μg mL−1; Fluka) and analyzed with a guava easyCyte 8 HT flow cytometer (Millipore) and InCyte software. The obtained readings were further analyzed with FlowJo software (TreeStar).
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