MDSCs were isolated from Trem1+/+ or Trem1–/– tumor-bearing mice as described earlier. ROS were detected using the ROS assay Kit (Invitrogen) per the manufacturer’s instructions. For induced activation experiments, MDSCs were cocultured with 30 ng/mL PMA (Thermo Fisher Scientific, 50-058-20001). ROS formation was acquired on an Attune NxT Acoustic Focusing flow cytometry platform (Thermo Fisher Scientific) and analyzed using FlowJo v10.0.
Ros assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The ROS assay kit is a laboratory instrument designed to measure the levels of reactive oxygen species (ROS) in cell samples. The kit provides a quantitative assessment of ROS, which are important signaling molecules and potential indicators of oxidative stress within cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa flow cytometer, by BD (3 mentions)
Ferulic acid, by Chengdu Herbpurify (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Attune nxt acoustic focusing flow cytometry platform, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Synergy h4, by Agilent Technologies (1 mentions)
Sod assay kit, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Facsdiva 9, by BD (1 mentions)
Software version 10, by FlowJo (1 mentions)
Ammonium chloride potassium ack lysing buffer, by Lonza (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ack lysing buffer, by Lonza (1 mentions)
Facsuite software, by BD (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Goat anti rabbit igg, by LI COR (1 mentions)
Annexin 5 apc 7 aad apoptosis detection kit, by BD (1 mentions)
24 protocols using ros assay kit
1
Measuring ROS in MDSCs from Trem1 Mice
2
Quantifying Cellular Oxidative Stress
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GASMCs (4×106 cells/ml) were seeded into 75 mm plates. After the cells were treated with reagents as aforementioned for 48 h, the cells were digested using 0.25% trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) for 15 min. Cells were resuspended in PBS following centrifugation at 1,000 × g at room temperature for 3 min. A Total Reactive Oxygen Species (ROS) Assay kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used, according to the manufacturer's protocol. Cells were analyzed using a flow cytometer (Invitrogen; Thermo Fisher Scientific, Inc.) and analysis software (FlowJo version 10.0; BD Biosciences) to determine the level of fluorescence.
3
Intracellular ROS Measurement in HUVECs
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HUVECs were treated by either UA (20 mg/dL) or normal saline for 24 h. Then cells were loaded with 5 μM working solution at 37 °C for 10 min and washed twice with warm buffer according to the experimental protocol of ROS assay kit (Invitrogen). After another 10 min incubation, cells were analyzed with a fluorescence microscope (PerkinElmer, US).
4
Measuring Hippocampus and Cortex ROS Levels
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ROS levels were measured by Invitrogen ROS Assay Kit (Shanghai, China) according to protocols provided by the company. Briefly, hippocampus and cortex tissues were homogenized on ice using 1% Triton-100 in phosphate-buffer (pH7.4) with protease inhibitors (1μg /ml pepstatin A, 1μg /ml leupeptin, and1μg /ml aprotinin). Lysates were centrifuged at 10 300g for 5 min and then the supernatant was collected.
The total protein content was determined by BCA kit. 50 μl sample (triplicate) and 50 μl catalyst solution were added to a black 96-well plate, and then the mixture was incubated for 5 min at room temperature. Then 100 μl dichlorodihydrofluorescein (DCFH) solution was added to the samples, and the mixture was sequentially incubated for 20 min at room temperature. Finally, the fluorescence intensity was measured on a microplate reader (Synergy H4, Bio-Tek, Winooski, VT, USA) at excitation 480 nm and emission 530 nm.
The total protein content was determined by BCA kit. 50 μl sample (triplicate) and 50 μl catalyst solution were added to a black 96-well plate, and then the mixture was incubated for 5 min at room temperature. Then 100 μl dichlorodihydrofluorescein (DCFH) solution was added to the samples, and the mixture was sequentially incubated for 20 min at room temperature. Finally, the fluorescence intensity was measured on a microplate reader (Synergy H4, Bio-Tek, Winooski, VT, USA) at excitation 480 nm and emission 530 nm.
5
Cytotoxicity and Oxidative Stress Assays
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SK-N-SH and SK-N-AS cells were collected and lysed with 0.2% Triton X-100 (Sigma-Aldrich). After centrifugation, 50 µL of the supernatant was collected for the next experiments. Lactate dehydrogenase (LDH) Cytotoxicity Detection Kit (Thermo Fisher Scientific) was used to assess the intracellular LDH level. Similarly, the activity levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were checked using the SOD Assay Kit or Cell MDA assay kit (Invitrogen). In addition, reactive oxygen species (ROS) generation was assessed with ROS Assay Kit (Invitrogen) referring to the recommended protocol.
6
Evaluating ROS Levels in ErbB2-Targeted Cells
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The ROS level in NCI-N87 and NCI-N87-TraRT cells was evaluated with total reactive oxygen species (ROS) assay kit (eBioscience). NCI-N87 and NCI-N87-TraRT cells were seeded in a flat-bottomed 24-well plate. Next day, cells were treated with indicated anti-ErbB2 mAbs or the mAb combination for indicated time. Then, cells were harvested and incubated with ROS assay stain for 60 min at 37°C. Then, the fluorescence of the cells was measured with FACSCalibur (Becton Dickinson) using excitation wavelength of 488 nm and emission wavelength of 520 nm.
7
ROS Production Quantification in Breast Cancer
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To evaluate the production of total reactive oxygen species (ROS), the ROS Assay Kit (eBioscience Inc., Affymetrix, San Diego, CA, USA) was utilized. Briefly, breast cancer cells at a density of 1×106 per T25 flask were treated with 3, 10, and 30 µM Artonin E for 24 h and trypsinized and centrifuged at 2,000 rpm for 5 minutes. The cells were resuspended in PBS and incubated in ROS (100 µL) assay stain in buffer solution at 37°C for 60 minutes before the flow cytometric analysis.
8
Intracellular ROS Quantification
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By utilizing the total reactive oxygen species (ROS) assay kit (Ebioscience, San Diego, CA, USA). The production of intracellular reactive oxygen species (ROS) was ascertained. The experiment was carried out by following the instructions of the manufacturer. In six-well plates, the cells were seeded at (1 × 106 cells/well) and then incubated for 24 h. After incubation, the cells were treated with varying concentrations of DEN or DEN-HPβCD complex for 24 h. Then, by using trypsin, the cells were detached. The detached cells were washed with PBS. Thereafter, the cells were treated with 100 µL ROS assay stain solution and incubated for 60 min at a temperature of 37 °C and CO2 presence of 5%. The BD face flow cytometer was then used for further analysis.
9
Quantifying Oxidative Stress in Leukocytes
10
Maternal-Fetal Oxidative Stress Assay
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Fifty μL of maternal peripheral blood and cord blood were stimulated with 50 μL of ROS assay mix containing 1:250 of ROS assay stain and ROS assay buffer [both from the ROS assay Kit (eBioscience, San Diego, CA, USA)], and 1 μL of phorbol myristate acetate (PMA; 3 μg/mL) (Millipore Sigma, Burlington, MA, USA). The unstimulated group received 1:250 ROS assay mix and 1X phosphate buffered saline (PBS) (Thermo Fisher Scientific/Gibco, Grand Island, NY, USA). The cells were incubated at 37°C with 5% CO2 for 60 min. Following incubation, erythrocytes were lysed using Ammonium-Chloride-Potassium (ACK) lysing buffer (Lonza, Walkersville, MD, USA), and the resulting leukocytes were collected after centrifugation at 300 x g for 5 min. Next, leukocytes were resuspended in 0.5 mL of 1X PBS and acquired using the BD LSRFortessa flow cytometer and FACSDiva 6.0 software to measure ROS production by neutrophils and monocytes. The analysis and figures were performed using the FlowJo software version 10.
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