Intracellular ROS detection was performed using a reactive oxygen detection kit (Beyotime, China) by flow cytometry. The cells were seeded in the 6-well plates with 4×105 cells per well and cultured for 24 hours. The cells were then treated with HNK, DSF/Cu, free combo drugs, LIPO, and CDX-LIPO for 12 hours. After wash, the cells were collected for ROS measurement.
Reactive oxygen detection kit
Manufactured by Beyotime
Sourced in China
The Reactive Oxygen Detection Kit is a laboratory tool designed to detect and quantify the presence of reactive oxygen species (ROS) in biological samples. The kit provides a standardized and reliable method for measuring ROS levels, which are important indicators of cellular stress and oxidative processes.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Mitochondrial membrane potential detection kit, by Beyotime (2 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Solarbio (2 mentions)
Seahorse xf glycolysis stress test kit, by Agilent Technologies (2 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Solarbio (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Riboflavin, by Merck Group (1 mentions)
Alendronate sodium, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Bca protein concentration determination kit, by Beyotime (1 mentions)
Tris 2 2 bipyridine ruthenium chloride, by Merck Group (1 mentions)
Rios origin water purification system, by Merck Group (1 mentions)
Cerium 3 nitrate hexahydrate, by Merck Group (1 mentions)
Rhodamine b, by Merck Group (1 mentions)
2 methylimidazole, by Merck Group (1 mentions)
Deionized water, by Merck Group (1 mentions)
Copper 2 nitrate hydrate, by Merck Group (1 mentions)
Ir 808, by Merck Group (1 mentions)
Tris hcl, by Beyotime (1 mentions)
Ir783, by Merck Group (1 mentions)
11 protocols using reactive oxygen detection kit
1
Intracellular ROS Detection by Flow Cytometry
2
Quantifying Intracellular Zinc and ROS
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Zinc ions concentration and ROS levels were detected using Zinpyr-1 (ChemCruz, sc-213,182) and Reactive Oxygen Detection Kit (Beyotime, S0033M-1). Zinpyr-1 (10 µM) and ROS probe (10 µM) were incubate with samples for 30 min at 37 °C, followed by three washes with M 2 medium. The fluorescent signals were detected using a confocal microscope.
3
Liposomal Drug Uptake and Apoptosis
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The tumor cells were incubated with the liposomes (equal to 4 μg/mL of drugs) for 24 h, and subsequently, caspase-3, cleaved-caspase-3, or RIPA levels were examined using Western blotting. The expression of EGFR/Erk/Akt and their phosphorrylated forms, and the levels of NOX3/MrsA/GPX4/Bcl-2 were also measured by Western blotting after 48-h treatment.
The tumor cells were treated with the liposomes (equal to 4 μg/mL each) for 24 h, and the intracellular ROS production was determined using a reactive oxygen detection kit (Beyotime, Shanghai, China).
The tumor cells were treated with the liposomes (equal to 4 μg/mL each) for 24 h, and the intracellular ROS production was determined using a reactive oxygen detection kit (Beyotime, Shanghai, China).
4
Cerium-Based Multifunctional Nanoplatform
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Cerium(III) nitrate hexahydrate (Ce(NO3)3·6H2O), alendronate sodium (AL), Tris(2,2-bipyridine) ruthenium chloride (RDPP), 2-methylimidazole (HMIM), riboflavin, rhodamine B, H2O2 (technical grade, 30%), LPS, and ORO were provided by Sigma–Aldrich (USA). Zoledronic acid hydrate (ZOL) was obtained from Damas-beta. PB and nitrotetrazolium blue chloride (NBT) were purchased from TCI (Japan). The CCK-8 kit was purchased from MCE (USA). oxLDL was purchased from Yiyuan Biotech (China). Chlorin e6 (Ce6) was provided by Frontier Scientific. A BCA protein concentration determination kit and a reactive oxygen detection kit were obtained from Beyotime Biotechnology. Penicillin–streptomycin solution (100X), fetal bovine serum (FBS), trypsin, Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 medium (1640) were obtained from Gibco (USA). High-purity (Milli-Q) water with a resistivity of 18.2 MΩ cm was obtained from an inline Millipore RiOs/Origin water purification system.
5
Photosensitizer-Mediated Cancer Therapy
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Copper(II) nitrate hydrate (Cu(NO3)2·3H2O), 2,5-dimercaptoterephthalic acid (DCA), IR 783, IR 808, Chlorin e6 (Ce6), Indocyanine green (ICG), Tris(2,2-bipyridine) ruthenium chloride (RDPP), H2O2 (technical grade, 30%) were provided by Sigma–Aldrich (USA). Nitrotetrazolium blue chloride (NBT) were purchased from TCI (Japan). The CCK-8 kit was purchasedfrom MCE (USA). A Tris-HCl (pH = 8.8, 1 M), anhydrous (NaCO3), BCA protein concentration determination kit, Calcein/PI cell viability/cytotoxicity assay kit, reactive oxygen detection kit, crystal violet were obtained from Beyotime (China). Matrigel® Matrix was purchased from Corning (USA). Penicillin–streptomycin solution (100X), foetal bovine serum (FBS), trypsin, Dulbecco’s modified Eagle’s447 medium (DMEM) were obtained from Gibco (USA). Phosphate-buffered saline (PBS, pH = 7.4) solution was prepared in the laboratory. All chemicals and reagents were of analytical grade and used as received without further purification. Millipore deionized water (18.2 MΩ · cm) was used for all experiments.
6
Doxorubicin-induced Cardiomyocyte Apoptosis
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Rat embryo cardiomyocyte H9c2 cultures were sourced from the Chinese Academy of Sciences Cell Bank (Beijing, China). RPMI-1640 medium and fetal bovine serum (FBS) both from HyClone Laboratories (Logan, UT, USA). Cleaved caspase-3, cleaved PARP, anti-GAPDH antibody and HRP secondary antibody all from Proteintech (Wuhan, China). Bmi-1 siRNA and NC siRNA both from from Dharmacon, Abgene Ltd. (Epsom, UK). Lipofectamine RNAiMAX was sourced from Invitrogen Corp. (Carlsbad, CA, USA). A BCA protein concentration assay kit, an Annexin V-FITC apoptosis detection kit and a reactive oxygen detection kit all from Beyotime Biotechnology (Nantong, China). DOX and esculetin both from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China).
7
Evaluating Immune Cell Activation and Metabolism
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Purified anti-mouse CD28 mAb, anti-mouse CD3 mAb, FITC-conjugated anti-mouse CD4 and PE-conjugated anti-mouse CD69 were purchased from BioLegend (San Diego, CA, USA). The Seahorse XF Glycolysis Stress Test Kit and Mitochondrial Stress Test Kit were provided by Agilent Technologies (Palo Alto, Calif.). Roswell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS) and penicillin/streptomycin were ordered from Gibco (South Logan, UT, USA). DAPI (4′,6-diamidino-2-phenylindole) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) were purchased from Solarbio (Beijing, China). Enhanced ATP Assay Kit, Mitochondrial Membrane Potential Detection Kit and Reactive Oxygen Detection Kit were supplied by Beyotime Biotechnology (Shanghai, China).
8
Intracellular ROS Detection by DCFH-DA
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Intracellular ROS was determined using the reactive oxygen detection kit (Beyotime, China). Briefly, cells were collected after transfection, and 2’-7’-dichlorofluorescein diacetate (DCFH-DA, 10 µM) was added to a serum-free medium and incubated at 37℃ for 20 min. After washing the cells three times with phosphate buffered saline (PBS), ROS content was determined using a flow cytometer (ACEA Biosciences, USA).
9
Inhibition of Ferroptosis and Oxidative Stress
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The methyltransferase G9a inhibitor DCG066 was a gift from Dr. Lanlan Zang of our central laboratory, and Fer-1 and Erastin were purchased from MedChemExpress (State of New Jersey, United States). RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Carsbad, CA, United States)and milk powder, penicillin/streptomycin, phosphate buffered saline (PBS), Cell Iron Assay Kit, GSH Assay Kit and MDA Assay Kit were purchased from Solarbio (Beijing, China). PVDF membrane and chemiluminescent solution were purchased from Millipore (Commonwealth of Massachusetts, United States). Total protein extraction kit, MTT assay kit, Calcein-AM/PI kit were purchased from Bestbio (Shanghai, China). Reactive Oxygen Detection Kit was purchased from Beyotime (Shanghai, China).
Antibodies for WB included GPX4 rabbit mAb (Lot.# 78p5366; WB 1:500), SCL7A11 rabbit mAb (Lot.# 84a5792; WB 1:500), Nrf2 rabbit mAb (Lot.# 65m9929; WB 1:500) and HO-1 rabbit mAb (Lot.# 80w1044; WB 1:500), were purchased from A nity Biosciences (Jiangsu, China); GAPDH mouse mAb (No.60004-1-Ig; WB 1:3000) and G9a rabbit mAb (Lot No: 800 vial; WB 1:500) purchased from A nity Biosciences (Wuhan, China) and Cell Signaling Technology Inc. (Danvers, United States).
Antibodies for WB included GPX4 rabbit mAb (Lot.# 78p5366; WB 1:500), SCL7A11 rabbit mAb (Lot.# 84a5792; WB 1:500), Nrf2 rabbit mAb (Lot.# 65m9929; WB 1:500) and HO-1 rabbit mAb (Lot.# 80w1044; WB 1:500), were purchased from A nity Biosciences (Jiangsu, China); GAPDH mouse mAb (No.60004-1-Ig; WB 1:3000) and G9a rabbit mAb (Lot No: 800 vial; WB 1:500) purchased from A nity Biosciences (Wuhan, China) and Cell Signaling Technology Inc. (Danvers, United States).
10
ROS Measurement in LoVo-induced MDSCs
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ROS was measured with the Reactive oxygen detection kit (Beyotime, Jiangsu, China). LoVo-induced MDSCs (1 × 105) were seeded in 96-well plates and the probe (DCFH-DA) was loaded into the cells according to the manufacturer's instructions. Then the DCFH-DA loaded MDSCs were cultured in the cell incubator for 30 min and washed 3 times with serum-free medium to eliminate the residual probe. The recombination proteins and specific inhibitors were added to the labeled MDSCs for 30 min, then the fluorescence was measured by a fluorescence microplate reader.
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