The preparation of the substrate cells was performed as previously described [4 (link)]. Briefly, adenocarcinoma human alveolar basal epithelial A549 cells were exposed to either photosensitizer 1 μM Ro 19-8022 (Hoffmann La Roche, Basel, Switzerland) or PBS (representing the negative control). After washing, the cells were exposed to light from a 500 W tungsten halogen lamp for 5 min on ice at a distance of 33 cm. Next, cells were washed, harvested through trypsinization, and counted. The cells were diluted to a concentration of 2 × 105 cells/mL, using freezing medium (50% RPMI, 40% FBS, 10% DMSO) (purchased from Gibco via Fisher Scientific, Landsmeer, The Netherlands; Sigma-Aldrich, Zwijndrecht, The Netherlands; Merck, Darmstadt, Germany). The resulting substrate cells were slowly frozen with the freezing container Mr. Frosty (Thermo Fisher Scientific, Zwijndrecht, The Netherlands) and stored at −80 °C until further use.
Ro 19 8022
Manufactured by Roche
Sourced in Switzerland
Ro 19-8022 is a laboratory equipment product developed by Roche. It serves as a core analytical tool for researchers and scientists. The detailed technical specifications and intended use of this product are not available for this response.
Automatically generated - may contain errors
Lab products found in correlation
Mr frosty, by Thermo Fisher Scientific (1 mentions)
Methyl methanesulfonate, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Etoposide, by Merck Group (1 mentions)
Sodium selenite, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Gentamycin sulfate, by Merck Group (1 mentions)
Iprodione, by Merck Group (1 mentions)
Cyprodinil, by Merck Group (1 mentions)
Hydromycin, by Thermo Fisher Scientific (1 mentions)
Procymidone, by Merck Group (1 mentions)
Fludioxonil, by Merck Group (1 mentions)
6 protocols using ro 19 8022
1
Preparation and Cryopreservation of Photosensitized A549 Cells
2
Oxidative Stress Induction in Cell Lines
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PBMCs and TK-6 cells were suspended in RMPI-1640 with 10% fetal bovine serum and placed in Petri dishes at a concentration of 2.5 × 105 cells/ml. 1 ml of cell suspension was used as control, and the remaining cells in the Petri dish were placed on ice and subsequently treated by adding photosensitizer Ro 19–8022 (a gift from F. Hoffmann-La Roche) at 1 µM and exposing to visible light (33 cm from a 500 W tungsten halogen source) for 5 min. Alternatively, cells were incubated with 10 mM KBrO3 in appropriate cell medium for 1 h at 37 °C. After treatment, cells were washed with PBS, centrifuged (250 × g, 5 min at 4 °C), and resuspended in 1 ml sterile PBS.
Adherent cells were seeded into 24–well plates and allowed to grow to 70–85% confluence. Samples were treated with KBrO3 or incubated with Ro 19–8022 plus light as above. After treatment, cells were detached with trypsin–EDTA, washed with PBS, centrifuged (250 × g, 5 min at 4 °C) and resuspended in 1 ml sterile PBS.
Adherent cells were seeded into 24–well plates and allowed to grow to 70–85% confluence. Samples were treated with KBrO3 or incubated with Ro 19–8022 plus light as above. After treatment, cells were detached with trypsin–EDTA, washed with PBS, centrifuged (250 × g, 5 min at 4 °C) and resuspended in 1 ml sterile PBS.
3
Fpg-Comet Assay DNA Oxidation Induction
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To induce DNA base oxidations detectable with the Fpg-enzyme (gift from Serge Boiteux, CNRS, France), defrosted PBMC (120 000 cells/mL) were placed on ice and treated with the compound Ro 19-8022 (Gift from Hoffman Laroche Ltd) (at 1 µM in PBS) during 2 min 30 sec under visible light (1000 W-halogen). Ro 19-8022 plus visible light exposure is an appropriate positive control for the Fpg-modified comet assay (Collins 2014) . Cells were then pelleted for 10 min at 200xg at 4°C.
4
Measuring DNA Strand Breaks Using Comet Assay
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DNA strand breaks were measured as previously described [23 (link)]. Strand breaks were visually scored and assigned to one of five classes in a blinded fashion as described by [23 (link)]. Cells treated with Ro19-8022 (gift from F. Hoffmann-La Roche, Basel, Switzerland) and white light were used as controls. The level of DNA damage was expressed as a total score calculated as:
5
HNE Impact on Cellular Metabolism and DNA Damage
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The 4-hydroxynonenal (HNE) was provided by Clinisciences (Nanterre, France). Fetal Bovine Serum (FBS) provided by ThermoFisher (Eindhoven, Netherlands). Puromycin, hydromycin and zeocin were provided by Fisher Scientific(Hampton, NH, USA). Epidermal growth factor (EGF), hydrocortisone, insulin, transferrin, sodium selenite (5 nM) and gentamycin sulfate (5 µg/mL), DMSO (dimethylsulfoxyde), procymidone, iprodione, cyprodinil, fludioxonil, lambda-cyhalothrin, methyl methanesulfonate (MMS), Trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), rotenone, oligomycin and etoposide were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). Ro 19-8022 was a gift from Hoffman Laroche Ltd. (Basel, Switzerland), and formamidopyrimidine-DNA glycosylase (Fpg) a gift from Serge Boiteux, CNRS, France.
6
Telomeric 8-oxoguanine Generation and Inhibition
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8-oxoguanine generation at telomeres: 100,000 cells were plated on coverslips in 35 mm dishes. 48 h post transfection, cells were incubated with 100 nM MG-2I dye for 15 min at 37 °C, 5% oxygen in phenol red-free DMEM. Cells were then exposed to 660 nm light (100 mW/cm2) for 10 min (unless specified otherwise) to induce the production of singlet oxygen. Cells were pretreated with transcription inhibitors for 90 min: α-amanitin (Sigma #A2263) and Cdk7 Inhibitor VIII, THZ1-Calbiochem (Sigma# 5323720001). Cells were fixed or harvested for further experiments.
Global 8-oxoguanine generation: Photosensitizer Ro 19-8022 was used to generate oxidative DNA damage (a kind gift from F. Hoffmann-La Roche, Ltd). Microscopic settings are explained in a separate section below. The following inhibitors were used: NEDD8 neddylation activating enzyme inhibitor (NAE1 inhibitor, MLN4924, Boston Biochem) and CSN5-catalysed cullin de-NEDDylation inhibitor (CSN5 inhibitor, SB-58-SN29, kindly provided by Novartis)84 (link).
Global 8-oxoguanine generation: Photosensitizer Ro 19-8022 was used to generate oxidative DNA damage (a kind gift from F. Hoffmann-La Roche, Ltd). Microscopic settings are explained in a separate section below. The following inhibitors were used: NEDD8 neddylation activating enzyme inhibitor (NAE1 inhibitor, MLN4924, Boston Biochem) and CSN5-catalysed cullin de-NEDDylation inhibitor (CSN5 inhibitor, SB-58-SN29, kindly provided by Novartis)84 (link).
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