Curcumin (95%) was purchased from Alfa Aesar (Kandel, Germany). PLGA (Purasorb PDLG 5002A) was obtained from Corbion Purac (Amsterdam, the Netherlands). The 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were ordered from Avanti Polar Lipids (Alabaster, AL, USA). The 2′,7′-dichlorofluorescein diacetate, tetramethylrhodamine ethyl ester (TMRE), and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) were purchased from Abcam (Cambridge, UK). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), poly(vinyl alcohol) (PVA, Mowiol 4–88), tert-butyl hydroperoxide (TBHP), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), and acetonitrile were all supplied by Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Acetone was acquired from Fisher Scientific GmbH (Dreieich, Germany). Ultrapure water (PURELAB flex 4, ELGA LabWater, High Wycombe, UK) was used in all experiments.
2 7 dichlorofluorescein diacetate
Manufactured by Abcam
Sourced in United Kingdom, United States
2′,7′-dichlorofluorescein diacetate is a cell-permeant fluorogenic dye that is used to measure intracellular oxidative stress and redox status. Upon entering cells, the diacetate form is cleaved by intracellular esterases, trapping the fluorescent 2′,7′-dichlorofluorescein inside the cell.
Automatically generated - may contain errors
Lab products found in correlation
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Curcumin, by Thermo Fisher Scientific (1 mentions)
Mowiol 4 88, by Merck Group (1 mentions)
2 7 dichlorofluorescein diacetate, by Merck Group (1 mentions)
Tert butyl hydroperoxide, by Merck Group (1 mentions)
Tetramethylrhodamine ethyl ester, by Abcam (1 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Abcam (1 mentions)
Purelab flex 4, by Elga LabWater (1 mentions)
Cellfix, by BD (1 mentions)
Phorbol myristate, by Merck Group (1 mentions)
2 7 dichlorofluorescein, by Molecular Devices (1 mentions)
4 protocols using 2 7 dichlorofluorescein diacetate
1
Curcumin-loaded PLGA-DPPC-PEG Nanoparticles
2
Measuring Endothelial ROS Production
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The production of reactive oxygen species (ROS) in endothelial cells was measured by using 2′,7′-dichlorofluorescein diacetate (Abcam, Cambridge, UK) as previously described.23 (link) In brief, HUVECs or HPMECs (2 × 105) were cultured in a 24-well plate overnight. The next day, medium was replaced with SFM containing 2′,7′-dichlorofluorescein diacetate (1.5 mL, 10 mmol/L) and subsequently stimulated as described earlier. Phorbol myristate (320 nmol/L; Sigma-Aldrich) was used as a positive control. The cells were detached and fixed in CELLFIX (250 µL, 1:10 dilution; Becton Dickinson, Heidelberg, Germany). ROS was measured by using flow cytometry. In some experiments before incubation with FITC-labeled mAbs, endothelial cells were treated with deglycosylated mAbs against FCGR (I, II, and III).
3
ROS Measurement in Cell Subsets
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The cultured cells were collected and stained with 2’,7’‐dichlorofluorescein diacetate (Abcam, Cambridge, MA, USA).[52] After incubation for 30 min at 37 °C, the fluorescence intensity of the ROS signal in different cell subsets was determined using flow cytometry. In some immunofluorescence experiments, the tumor tissue samples were stained with a ROS probe.
4
Measurement of Lung ROS Production
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ROS production was detected as previously reported with slight modifications [24 (link),25 (link)]. Briefly, lung homogenate (10 μL) was incubated with 10 μM of 2′,7′-dichlorofluorescein diacetate (Abcam; Cambridge, MN, USA) at 37 °C for 0.5 h, and the fluorescence intensity emitted from 2′,7′-dichlorofluorescein was monitored at an excitation wavelength of 490 nm and an emission of 520 nm using a VersaMaxTM fluorescence reader (Molecular Devices; Sunnyvale, CA, USA).
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