ROS generation was determined using the ROS kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, SH-SY5Y cells were seeded in 96-well black plates with clear bottoms at a density 1 × 105 cells/mL. Cells were pretreated with CC or FCC for 2 h followed by co-culture with H2O2 for 3 h. The medium was removed and washed by Hanks’ Balanced Salt Solution (HBSS). The master reaction mix (100 µL) was added to the wells and then cells were incubated in 5% CO2, 37 °C incubator for 30 min. The fluorescence intensity was measured at an excitation of 640 nm and an emission of 675 nm.
Ros kit
Manufactured by Merck Group
Sourced in United States
The ROS kit is a laboratory equipment product designed for the detection and measurement of reactive oxygen species (ROS) in biological samples. It provides the necessary tools and reagents for researchers to quantify the presence of various ROS, such as superoxide, hydrogen peroxide, and hydroxyl radicals, within their experiments. The core function of the ROS kit is to enable accurate and reliable ROS analysis, supporting various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dfc450 c camera, by Leica (1 mentions)
Las af ver 3, by Leica (1 mentions)
Model dmi8, by Leica (1 mentions)
Live dead kit, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Tissue mitochondria isolation kit, by Beyotime (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
5 protocols using ros kit
1
ROS Determination in SH-SY5Y Cells
2
Fluorescence Analysis of Cell Death and Proliferation
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Signals emitted in the Live/Dead Kit (Sigma-Aldrich 04511) and ROS Kit (Sigma-Aldrich MAK 143) assays used to examine the mechanisms of cell death and proliferation were analyzed in an inverted epifluorescence microscope (Model DMi8 , Leica Microsystems, Germany) equipped with fluorescence filters (546/10 RHOD excitation filter and 585/40 emission, DAPI 350/50 excitation filter and 460/40 emission, and FITC excitation filter 480/40 and emission 527/30), a monochrome DFC 450C camera (Leica Microsystems) and fluorescence overlay software (LAS AF ver. 3.1.0, Leica Microsystems CMS GmbH). Since the ommochromes are capable of absorbing ultraviolet rays and can interact with ultraviolet light, the fluorescence emission in the suspension of methanol/HCl pigments (0.31-10 mg/ml) in nutrient broth medium (pH 7.4) was evaluated.
3
Quantifying Cellular Reactive Oxygen Species
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ROS was quantified using a ROS kit (Sigma) according to the manufacturer’s instructions. Cellular ROS levels were measured by flow cytometry. Briefly, 2 × 105 cells were plated onto six-well cell culture plates and allowed to attach to the wells overnight. Thereafter, adhered cells were treated with PL (1.0, 2.0, or 4.0 μM) in the presence or absence of NAC (5 mM) pre-treatment for 2 h. After removal of the medium, the ROS indicator, DCFH-DA (10 μM), was added to fresh FBS-free medium and incubated for 30 min at 37 °C in the dark. Cells were then collected, and fluorescence was analyzed using a FACS Calibur flow cytometer (BD Biosciences, CA).
4
Zebrafish Intestinal Mitochondria Isolation and ROS Measurement
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Intestinal mitochondria were isolated by using a tissue mitochondria isolation kit (Beyotime Biotechnology, Shanghai, China). In brief, fresh intestine was collected from zebrafish and homogenized for 10 times in the isolation buffer using a glass homogenizer. The homogenate was then centrifugated at 1000 g for 10 min. The supernatant was then aspirated and further centrifugated at 11,000 g for 10 min. The precipitation was resuspended in the mitochondria storage buffer. The purity of isolated mitochondria was assessed according to Fernandez-Vizarra et al. (2006) (Figures S9 A–S9D). Then isolated mitochondria were incubated with a fluorescent probe provided by the ROS kit (Sigma, USA) for 2 h in a 96-well flat-bottom plate. The fluorescence intensity was measured excitation 490 nm and emission 520 nm. The ROS level was expressed as the fold change compared with the HFD group. Rosup was applied as a positive control (Figure S9 E).
5
Intracellular ROS Measurement in Cancer Cells
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The non-small cell lung cancer (NSCLC) cell line A549, pancreatic cancer cell line PANC and human normal hepatocyte line MIHA were obtained from the Cell Resource Center of Peking Union Medical College. Cells were cultured in DMEM (Gibco, USA) at 37 °C under 5% CO 2 supplemented with 10% fetal calf serum (Gibco, USA) and 100 U/mL penicillin and streptomycin (HyClone, USA). Measurement of intracellular ROS production ROS were monitored using a ROS kit (Sigma, USA) according to the manufacturer's instructions. In short, 1.5 × 10 5 cells were grown in six-well cell culture plates and incubated overnight. After treatment with or without drugs, cells were incubated with 10 μM DCFH-DA for 30 min at 37 °C in the dark. DCF uorescence intensity was measured by ow cytometry (BD FACS Calibur, BD Biosciences, USA) and Nikon uorescence microscopy (Nikon, Japan).
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