2×105 cells /2ml were seeded into a 6-well plate (Corning) overnight, cells were pretreated with N-Acetyl-L-cysteine(NAC, sigma, A8199) or vehicle for 4h, then HI-TOPK-032 was added to the cells. After 6h cells were washed by PBS twice and loaded with 10mM general ROS indicatorCM-H2DCFDA (sigma) in serum free DMEM for 30 minutes at 37°C in the dark, then examined by fluorescence microscopy.
A8199
Manufactured by Merck Group
Sourced in United States
The A8199 is a laboratory equipment product manufactured by Merck Group. It is designed to perform core functions related to scientific research and analysis. The detailed specifications and intended use of this product are not included in this response to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
6 well plate, by Corning (1 mentions)
A8960, by Merck Group (1 mentions)
H3149, by Merck Group (1 mentions)
L3755, by Merck Group (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
Endothelial cell growth supplement (ecgs), by Merck Group (1 mentions)
Pt 5006, by Lonza (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
4 protocols using a8199
1
Cellular ROS Measurement Assay
2
Isolation and Cultivation of hADSC and HUVEC
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Human adipose‐derived stem cells (hADSC, PT‐5006, Clonetics, Lonza) were cultured in keratinocyte‐SFM (17005‐042, GIBCO‐Invitrogen), supplemented with 2 mmol/L N‐acetyl‐L‐cysteine (NAC, A8199, SIGMA), L‐ascorbic acid 2‐phosphate (Asc 2P, A8960, SIGMA) and 5% foetal bovine serum (16000044, GIBCO‐Invitrogen).
Human umbilical vein endothelial cells (HUVECs, BCRC No. H‐UV001) were cultured in medium 199, supplemented with 10% foetal bovine serum, 25 U/mL heparin (H‐3149, SIGMA), 30 µg/mL endothelial cell growth supplement (ECGS, 02‐102, Millipore), 2 mmol/L L‐glutamine, 1.5 g/L sodium bicarbonate and 1X penicillin/streptomycin.
For culture media collection, cell culture was limited to eight passages. 1 × 106 hADSC cells were cultured in 10 mL serum‐free media, with/without 1 μg/mL lipopolysaccharides (LPS, L3755, SIGMA), for 24 hours. The culture media (500 mL) were harvested and centrifuged at 300 g for 5 minutes to remove cells and cell debris. The supernatants were concentrated using the Amicon® Ultra‐15 (UFC900324, Merck‐Millipore) and transferred into new tubes for further use.
Human umbilical vein endothelial cells (HUVECs, BCRC No. H‐UV001) were cultured in medium 199, supplemented with 10% foetal bovine serum, 25 U/mL heparin (H‐3149, SIGMA), 30 µg/mL endothelial cell growth supplement (ECGS, 02‐102, Millipore), 2 mmol/L L‐glutamine, 1.5 g/L sodium bicarbonate and 1X penicillin/streptomycin.
For culture media collection, cell culture was limited to eight passages. 1 × 106 hADSC cells were cultured in 10 mL serum‐free media, with/without 1 μg/mL lipopolysaccharides (LPS, L3755, SIGMA), for 24 hours. The culture media (500 mL) were harvested and centrifuged at 300 g for 5 minutes to remove cells and cell debris. The supernatants were concentrated using the Amicon® Ultra‐15 (UFC900324, Merck‐Millipore) and transferred into new tubes for further use.
3
NAC Treatment of Cultured Cells
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1×104 cells were plated in 6 replicate wells in a 96-well dish. Twelve hours later, cells were treated with N-acetyl-L-cysteine (NAC) (2 mM)(A8199, Sigma) final concentration for a period of 1 hour.
4
NAC Toxicity and Protective Effects
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To determine the toxicity of NAC, 1 × 104 HK-2 cells were treated with various concentrations (100, 300, 600, and 1000 µg/mL) of NAC (A8199, Sigma-Aldrich, Saint Louis, USA) for 24 h. To determine the protective effect of NAC, 1 × 104 HK-2 cells were treated with 10 µM K2Cr2O7 and various concentrations of NAC (100, 300, 600, and 1000 µg/mL) at different time-points after chromium exposure (0, 1, 2, 4, and 8 h). After incubating further for 24 h, cell viabilities were directly examined by an inverted microscope, Eclipse Ti-U (Nikon, Tokyo, Japan), under 400× g magnification, and indirectly assayed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) kit (Sigma-Aldrich, Schnelldorf, Germany), according to the manufacturer’s instruction. The absorbance at A570 nm was determined by an ELISA reader (Multiskan EX, Labsystems, MA, USA).
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