Multimode microplate reader

Oxidative stress was induced on differentiated R28 cells using H₂O₂ (SigMA, USA) following the method of Song et al. [32 (link)] with some minor modifications. Briefly, R28 cells were treated with multiple concentrations of H₂O₂ (0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.5 mM) for 24 h and cellular cytotoxicity was measured. In order to determine the effect of FTY on H₂O₂-induced oxidative stress, cells were pre-treated with 25 nM FTY (as described previously) and changes in cytotoxicity was measured.
Lactate dehydrogenase (LDH) assay was used to determine cellular cytotoxicity. LDH released into the culture media from damaged cells was measured following the manufacturer’s instructions (CytoTox 96 non-radioactive cytotoxicity assay kit; Promega Corporation, Madison, WI, USA). The level of LDH release was normalized to the total LDH content following cell lysis in a medium. The absorbance was determined at 490 nm using a Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany). LDH release was expressed as a percentage of the maximum LDH released after cell lysis.

A blank was run for every batch of samples during spectrophotometry analysis, and duplicate testing was conducted for all the parameters in order to ensure quality. In order to minimize cross contamination across samples, collection containers were thoroughly cleaned and oven-dried prior to field visits. In addition, before collection, containers were rinsed with water from the same collection source. All parameters were analyzed under standard conditions and using standard methods of water analysis [28 –30 ]. All plastics used for analysis were rinsed twice with distilled water prior to usage. Measurements were conducted twice per sample for quality control, and the average concentration was reported. Calibration of the Multimode Microplate Reader (Berthold Technologies, Germany) is normally conducted every six months, while the calibration of the physicochemical analytical equipment (Table 1) is conducted before every measurement using standards or blanks, according to manufacturer’s instructions (HACH Turbidimeter 2100 p, HQ40D HACH Conductivity, pH, and DO Meter, and DR 2800 HACH Spectrophotometer–HACH Company, Colorado, United States). In addition, for the water quality parameter measurements, water samples were tested immediately upon arrival to the lab, while other tests were conducted within 24 hours of collection time.

IFN-y ELISA was performed using the human IFN-y DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions. Briefly, 96-well plates were coated overnight with capture antibody, washed twice in wash buffer then blocked with reagent diluent for 2 hrs at RT. 100 μl of PBMC culture supernatants were added and incubated for 1 hr at RT and washed twice in wash buffer. 100 μl detection antibody diluted in reagent diluent was added per well and incubated for 2 hrs at RT. Plates were washed twice in wash buffer. 100 μl streptavidin-HRP dilution was added to the plates and incubated for 20 minutes in the dark at RT, plates were washed twice in wash buffer. The reaction was developed using 200 μl substrate solution for 20 minutes in the dark at RT then stopped with 50 μl stop solution. Optical density was measured at 450 nm on a multimode microplate reader (Berthold). Serial dilutions of standard were run on each plate. Concentrations were calculated by linear regression of standard concentrations ranging 0–600 pg/ml and normalized to the number of stimulated PBMC. The assay sensitivity was 5 pg/ml.