Donors were submitted to treatment with and without compounds and incubated at 37°C for 5 h with shaking. After that, the ROS detection assay was done as described by Castro-Alférez et al.,27 (link) with adjustments. Measurements of chemical hydrolysis of the probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma–Aldrich, St. Louis, USA) in the fluorescent compound 2,7-dichlorofluorescein (DCF) were detected by fluorescence spectroscopy at 522 and 498 nm emission and excitation wavelengths, respectively. Fluorescence detection was performed in 96-well solid-bottom black plates using the TECAN 200 Pro (Tecan, Männedorf, Switzerland) fluorimeter. H2O2 at 2.5 mM was used as a positive control for the experiment.
200 pro
Manufactured by Tecan
Sourced in Switzerland, Germany
The TECAN 200 Pro is a liquid handling system designed for laboratory automation. It features precise pipetting, dilution, and sample transfer capabilities to support a variety of applications in research and diagnostic laboratories.
Automatically generated - may contain errors
2 protocols using 200 pro
1
Quantifying Oxidative Stress Responses
2
Quantification of LDH and Glucose Release
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To measure the amount of LDH and GLU released from the treated NR8383 cells, cell culture supernatants were harvested and centrifuged (10 min, 200 g) to remove cell debris. From each well, 50 μL was incubated with LDH reaction mix (Roche Cytotoxicity Kit; Roche, Germany) and evaluated as described by the manufacturer. Measurements were corrected for cell free-adsorption and normalized to the PC value (set to 100 %) obtained from NR8383 cells lysed with 0.1 % Triton X-100 (Sigma Aldrich, Germany). To measure GLU activity, 50 µL of the supernatant was incubated at 37 °C with 100 μL 0.2 M sodium acetate buffer (pH 5) containing 13.3 mM p-nitrophenyl-d -glucuronide (Sigma Aldrich, Germany) and 0.1 % Triton X-100 [50 (link)]. The reaction was terminated after 2 h by addition of 100 μL 0.2 M NaOH (Merck KGaA, Germany). Optical density (OD) was measured at 405 nm in a plate photometer (Tecan 200Pro, Tecan, Germany); values were corrected for cell free-adsorption and normalized to the PC value (set to 100 %) again obtained from NR8383 cells lysed with 0.1 % Triton X-100.
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