Intracellular ROS detection was carried out by dihydroethidium (DHE) staining followed by flow cytometry analysis. Immediately after UVA irradiation, cells were washed with PBS and 200 nM DHE (ThermoFisher) in PBS was added to each well. Cells were incubated 30 min at 37 °C and trypsinized. 500 μL complete DMEM was added to neutralize the trypsin-EDTA solution (Biosera). Samples were analyzed by a FACSCalibur flow cytometer (BD Biosciences) using CellQuestPro software (BD Biosciences), and fluorescent signal was measured in FL3.
Trypsin edta solution
Manufactured by Biosera
Sourced in France
Trypsin-EDTA solution is a cell culture reagent used for the dissociation and detachment of adherent cells from cell culture vessels. It contains the proteolytic enzyme trypsin and the chelating agent EDTA, which work together to disrupt cell-cell and cell-matrix interactions, allowing cells to be harvested for further experiments or passaging.
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2 protocols using trypsin edta solution
1
Intracellular ROS Detection by DHE Staining
2
Cell Line Culturing for MTT Assay
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Three human cell lines (MCF-7, A549, SKOV3) were obtained from the National Cell Bank of Iran (NCBI, Pasteur Institute, Tehran, I.R. Iran) and were cultured under aseptic conditions in the complete culture media containing Roswell Park Memorial Institute (RPMI) 1640 medium (Biosera, France), 10% fetal bovine serum (FBS; Gibco, USA), and 1% penicillin/streptomycin (Biosera, France) at 37 °C in a humidified CO2 incubator. Following 70-80 confluency, the cells were trypsinized using 25% trypsin-EDTA solution (Biosera, France), were counted and then seeded in 96-well cell culture microplate in a density of 10 × 103 cells per well in 100 μL complete culture medium for MTT assay.
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