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ROS 活性氧检测试剂盒
Reactive Oxygen Species Assay Kit （ROS）
Kit Contents：
|
Name |
Package |
|
|
Component A |
H2DCFDA（10mM）in DMSO |
0.1mL |
|
Component B |
Reactive oxygen control（Rosup, 50mg/mL） |
1mL |
Storage:
Component A: Store at -20°C protected from light, avoid multiple freeze/thaw cycles. Stable for12 months after received.
Component B: Store at -20°C protected from light. Warm to room temperature before use
Introduction：
The Reactive Oxygen Species (ROS) Detection Kit provides a simple, specific and highly sensitive method for the real-time measurement of ROS levels in living cells or antioxidant activity of test compounds. It utilizes H2DCFDA, a cell-permeable fluorogenic probe, compatible with phenol red, FBS and BSA to detect hydroxyl, peroxyl, or other reactive oxygen species in live cells. Upon the cell entry, H2DCFDA is modified by cellular esterases to form a non-fluorescent H2DCF which is oxidized by intracellular ROS to yield a highly fluorescent product that can be detected by using FACS, microplate reader, or fluorescence microscope (Ex/Em 495/529 nm). The fluorescence intensity is proportional to the ROS levels. Reactive oxygen species are chemically reactive oxygen- containing molecules that are generated as a natural byproduct of the oxygen metabolism. ROS are constantly generated and eliminated under normal physiologic conditions and have important functions in cell signaling, homeostasis, and clearance of microbial infections. During times of environmental stress such as exposure to UV or heat, or during infection, ROS levels can increase dramatically leading to oxidative stress. Oxidative stress can result in damage to cellular proteins, lipids, and DNA, and has been linked to cardiovascular disease, cancer, diabetes, inflammation, and aging. Examples of ROS include superoxide ions and peroxides..
ROS Detection Assay Protocol:
The protocol was developed using live bovine pulmonary artery endothelial cells (BPAEC) and MRC5 human lung fibroblasts adhering to coverslips, but is amenable for use with other cell types. An additional protocol is provided for the use of (Component B, Rosup) as a positive control for the induction of ROS, which, if desired, must be performed before labeling with H2DCFDA.
1. Labeling with H2DCFDA
1.1 The Component A (H2DCFDA（10mM）in DMSO) is thoroughly thawed at room temperature (about 25°C)
1.2 Prepare 25 μM carboxy-H2DCFDA working solution. Add 5.0 μL of the 10 mM H2DCFDA stock solution (prepared in step 1.1) to 2.0 mL of warm HBSS/Ca/Mg or other suitable buffer.
1.3 Wash cells. Gently wash cells once with warm HBSS/Ca/Mg or other suitable buffer.
1.4 Label cells. Apply a sufficient amount of the 25 μM H2DCFDA working solution (prepared in step 1.2) to cover the cells adhering to the coverslip(s). Incubate for 30 minutes at 37°C, protected from light.
1.5 Wash cells. Gently wash the coverslips three times in warm HBSS/Ca/Mg or other suitable buffer.
1.6 Mount in warm buffer and image immediately. Best results are obtained when imaging takes place immediately after washing and mounting the sample.
2. Induction of Cellular ROS Production with Component B(Rosup):
2.1 Make 100 μM working solution of Rosup.
Dilute the Component B 1:5000 in appropriate complete growth media to produce a 100 μM working solution. For example, add 1μL Component B to 5 mL of complete media. to make 5.0 mL of 100 μM Rosup working solution,
2.2 Induce ROS production in cells. Apply a sufficient amount of the 100 μM Rosup working solution (prepared in step 2.1) to the cells adhering to the coverslip(s). Incubate the coverslip(s)at 37°C and 5% CO2. During development of the product using BPAE and MRC5 cells, a 60–90 minute incubation period was required. Appropriate incubation periods for ROS production in other cell lines should be determined empirically. After induction, label the cells with H2DCFDA starting with step 1.1, above
2.3 Wash cells. Gently wash the coverslips twice in warm HBSS/ Ca/Mg or other suitable buffer. After washing, label the cells with H2DCFDA starting with step 1.1, above.
PROTOCOL FOR ADHERENT CELLS
NOTE: To create positive controls, oxidative activity is stimulated with Component B prior to analysis
1. Seed adherent cells at 25 x 103 per well one day before performing the assay.
2. Remove the media and add 100 μL/well of diluted Component A.
3. Remove Component A and stain cells by adding 100 μ/well of Probe Working Solution.
4. Incubate at cells’ optimal temperature in dark conditions. An incubation time of 15～60 minutes is enough.
5. Remove media and add at least 100μL of PBS. Measure fluorescence immediately.
PROTOCOL FOR SUSPENSION CELLS
NOTE: To create positive controls, oxidative activity is stimulated with Component B prior to analysis
1. Grow suspension cells in sufficient amount. (In the step 5 you will need 100 x 103 cells per group).
2. Collect and wash cells with PBS using centrifugation.
3. Resuspend cells at a density of 1x106 cells/mL. Stain the cells with the desired volume of Probe Working Solution.
4. Incubate at cells’ optimal temperature in dark conditions. An incubation time of 15–60 minutes is enough.
5. Wash cells by centrifugation. Resuspend cells in PBS, seed in 96-well microplate or fluorimeter with 100,000 stained cells/well and measure fluorescence immediately.
PROTOCOL FORFLOW CYTOMETER
NOTE: To create positive controls, oxidative activity is stimulated with Component B prior to analysis
1. Grow cells (adherent or suspension) so that on the day of the experiment there are at least 15 x 103 cells per assayed condition (treatment, dose, time). Include in the calculation enough cells for a control
2. Harvest cells and ensure a single cell suspension by gently pipetting up and down suspension cells or by fully detaching adherent cells (e.g. trypsinize and quench with media).
3. Stain cells in culture media with 10-25 μM DCFH-DA and incubate for 30 minutes at 37°C. Once the incubation is completed, DO NOT wash the cells
4. After staining, treat the cells with compound(s) of interest and ensure that appropriate controls are included. If using Rosup as positive control, optimal signal is obtained after 4 hours of treatment.
5. Analyze on flow cytometer. Establish forward and side scatter gates to exclude debris and cellular aggregates from analysis. DCF should be excited by the 488 nm laser and should be detected at 535 nm.
NOTE: Always wear lab coats, gloves and goggles when working with our products although they are low-risk chemicals for R&D only.
使用活性氧检测试剂盒(Reactive oxygen species assay kit)显示CHO细胞内活性氧荧光。
左图：CHO细胞用试剂盒配备的活性氧阳性对照处理；右图：正常CHO细胞。绿色荧光表明细胞活性氧急剧增加，并能显示其定位。
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Description
| Name | ROS, Reactive Oxygen Species Assay Kit | ||
|---|---|---|---|
| CAT# | 040-1000T | CAS# | N/A |
| Storage# | −20°C Sealed & desiccated & Minimized light exposure & Minimized Freezing And Thawing | Shelf Life# | 24 months |
| Ex(nm)# | N/A | Em(nm)# | N/A |
| MW# | N/A | Solvent# | |
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