Hemoglobin (Hb), 9,10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA), and bis(2,4,5-trichloro-6-carbopentoxyphenyl) oxalate (CPPO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lactate oxidase (LOX) was purchased from Shanghai Yuanye Bio-Technology (Shanghai, China). Chlorin e6 (Ce6) was purchased from J&K Scientific Ltd (Beijing, China). RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (AG, Switzerland). Rhodamine phalloidin was obtained from Invitrogen (USA). Trypsin-EDTA and penicillin-streptomycin, were purchased from Corning life sciences (Wujiang, China). Hoechst 33342, calcein-AM/propidium iodide (PI), Apoptosis Analysis Kit, and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Solarbio® life sciences (Beijing, China). Cell cycle Analysis Kit was obtained from Beyotime (Beijing, China). The antibodies and corresponding dilution ratio were listed in Supplementary Table 2 . The siRNA sequences (Sangon Biotech, Shanghai) were listed in Supplementary Table 3 . pcDNA3.1-MEF2C (KWC20220217AZGYQ-PC01) and pcDNA3.1-NAMPT (KWC20220217AZGYQ-PC02) were constructed by Hanbio Biotech (Shanghai, China).
2 7 dichlorofluorescin diacetate dcfh da
Manufactured by Solarbio
Sourced in China
2',7'-dichlorofluorescin diacetate (DCFH-DA) is a fluorogenic probe that can be used to detect and measure reactive oxygen species (ROS) in biological systems. It is a non-fluorescent compound that can be oxidized by ROS to produce a fluorescent compound, 2',7'-dichlorofluorescein (DCF), which can be detected by fluorescence spectroscopy.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Trypsin edta, by Corning (1 mentions)
Cell cycle analysis kit, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lactate oxidase lox, by Yuanye Bio-Technology (1 mentions)
Chlorin e6 ce6, by J&K Scientific (1 mentions)
Hoechst 33342, by Solarbio (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Gsh assay kit, by Beyotime (1 mentions)
Penicillin streptomycin, by Solarbio (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
2 2 azino bis 3 ethylbenzothiazoline 6 sulfonic acid diammonium salt abts, by Merck Group (1 mentions)
Caco 2 cell, by American Type Culture Collection (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Ferrozine, by Merck Group (1 mentions)
Trypsin, by Yuanye Bio-Technology (1 mentions)
Glucoamylase, by Yuanye Bio-Technology (1 mentions)
7 protocols using 2 7 dichlorofluorescin diacetate dcfh da
1
Comprehensive Cellular Assay Protocol
2
Measurement of Intracellular ROS using DCFH-DA
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The 2,7-dichlorofluorescin diacetate (DCFH-DA; Solarbio, Beijing, China) is a cell permeable peroxide-sensitive fluorescent probe used to detect intracellular free radicals. Once DCFH-DA enters into the cells and hydrolyses to dichlorofluorescin (DCFH) in the presence of free radicals such as H 2 O 2 , and peroxides, DCFH is oxidised to the green fluorescent product dichlorofluorescein (DCF) and trapped in the cell compartment, which can be measured by a fluorescence spectrophotometer. The wavelength of the laser was 488-525 nm (excitation and emission, respectively). In the present study, two GC groups were collected and incubated with DCFH-DA at 37°C for 20 minutes. Then, fluorescent DCF were measured using a fluorescence spectrophotometer, and the relative absorbance values that characterise the ROS content were measured. At 30 minutes of CCCP and BA exposure, inhibitor-treated GCs and control cells were collected and incubated with DCFH-DA at 37°C for 20 minutes. Then, the ROS content was measured using a ROS Detection Kit.
3
TLR-4 Modulation Impacts MDM ROS
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The TLR-4 on MDMs were pre-activated or pre-blocked with lipopolysaccharide (LPS; a TLR4-specific activator; Sigma-Aldrich) or 7E3 (a TLR-4-blocking antibody; Abcam, Cambridge, UK) for 24 h, and then rBsCPI-1 was added and incubated for 12 h. The fluorogenic dye 2′-7′dichlorofluorescin diacetate (DCFH-DA; Solarbio) was diluted 1:1000 with serum-free medium to a final concentration 10 μM/l. MDMs were incubated with DCFH-DA at 37 °C, 5% CO2 for 20 min, then washed three times with serum-free cell culture medium to sufficiently removed DCFH-DA that did not enter the cells. The cells were then collected and detected by measurement fluorescence at 488 nm excitation and 525 nm emission wavelengths.
MDMs were pre-incubated with 10 µM Nigericin or MCC950 sodium for 4 h. rBsCPI-1 was then added and the solution incubated for 12 h. ROS analysis experiments were then carried out.
MDMs were pre-incubated with 10 µM Nigericin or MCC950 sodium for 4 h. rBsCPI-1 was then added and the solution incubated for 12 h. ROS analysis experiments were then carried out.
4
Preparation of Biodegradable Polymer Materials
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Agarose was purchased from Yare Shanghai (Shanghai, China). PLGA and PLA were obtained from Chongqing Yusi medical technology cable Co., Ltd. (Chongqing, China). A GSH Assay Kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was obtained from Solarbio life sciences (Beijing, China). The other reagents used in this work were purchased from Sinopharm Chemical Reagent (Shanghai, China) and Aladdin-Reagent (Shanghai, China).
5
Broken Rice Bioactive Peptide Production
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Broken rice was supported by Jinhe Group Co., Ltd. (Herbin, China). Alkaline protease from Bacillus licheniformis, Neutral proteinase, Trypsin, α-amylase, and Glucoamylase were purchased from Yuanye Bio-Technology Co., Ltd (Shanghai, China). Pepsin and ChymoTrypsin were obtained from SUMMUS biological technology co., Ltd. (Harbin, China). DPPH, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and ferrozine were donated by Sigma Chemical Co. (St. Louis, MO, USA). The Caco-2 cells, derived from human colon adenocarcinoma, were purchased from the American Type Culture Collection (ATCC). Dulbecco′s modified Eagle medium (DMEM, high glucose), fetal bovine serum (FBS), penicillin-streptomycin, and 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). A Cell Counting Kit-8 (CCK-8) was obtained from Beyotime Biotechnology (Shanghai, China). All the other chemicals used in this study were analytical grade.
6
Aβ-Induced Neuronal Apoptosis Assay
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THS (≥99%), RES (≥99%), and PIC (≥99%) were obtained from J&K Scientific, Inc. (Beijing, China). Aβ25-35 (≥99%) was purchased from Chinese Peptide, Inc. (Beijing, China). Neurobasal, B27, 0.25% trypsin, sodium pyruvate, and 2-mercaptoethanol were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum was purchased from Hyclone Laboratory, Inc. (Logan, UT, USA). Furthermore, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was obtained from AMRESCO, Inc. (Solon, OH, USA). Also, 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The following rabbit antibodies were purchased from Cell Signalling Technology, Inc. (Boston, MA, USA): phosphor-Bad (Ser-136), phosphor-Akt (Ser-473), Akt, Bax, Bcl-2, caspase-8, cleaved caspase-3, cleaved caspase-9, and cleaved PARP. Other analytical grade regents were obtained from Beijing Chemical Reagent, Inc. (Beijing, China).
7
Intracellular ROS Quantification via DCFH-DA
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Intracellular ROS levels were evaluated using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (Solarbio, Beijing, China). In brief, the cells were incubated with 10 μmol/L DCFH-DA at 37°C for 20 min and then washed with PBS. Fluorescence was evaluated using a fluorescence microscope at excitation and emission wavelengths of 488 and 525 nm, respectively.
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