Measure it high sensitivity nitrite assay kit

Nitric oxide (NO) concentration was indirectly assessed by measuring the concentration of nitrite, a by-product of NO degradation, with a Measure-IT™ High-Sensitivity Nitrite Assay Kit according to the manufacturer’s directions (Invitrogen by Life Technologies, Grand Island, NY). Conditioned media collected from flow chambers and the anterior segment perfusions every 24 hours was used to measure nitrite concentration in the samples. Briefly, a standard curve was made using serial dilutions of a nitrite standard included in the kit. The samples and standards were combined with a working solution included in the kit and incubated at room temperature. The fluorescence of the nitrite-containing samples was measured at using a SpectraMax M5 plate reader coupled with SoftMax Pro 7 software (Molecular Devices, Sunnyvale, CA). Linear regression analysis using data from the standard curve was used to estimate the nitrite concentrations of the samples.

The supernatants from the control or infected MDSCs in vivo or cultured in vitro were removed from the cells and centrifuged at 500× g for 5 min to pellet the non-adherent cells. The amount of nitrite present in the supernatant was analyzed using the Measure-IT™ High-Sensitivity Nitrite Assay Kit (Invitrogen, Carlsbad, CA, USA). The supernatant samples (10 µL) or nitrite standards were assayed in triplicate following the manufacturer’s protocol. The fluorescence was quantified from the samples at an excitation/emission of 365/450 nm using a SpectraMax iD3 (Molecular Devices, San Jose, CA, USA).

After incubation with ECM, parasites were centrifuged (10,000 x G, 5 minutes) and the supernatant separated for nitric oxide quantification, as described by the manufacturer (Measure-iT High-Sensitivity Nitrite Assay Kit, Invitrogen)

Nitrite oxide (NO) production in cell culture supernatants was quantified using Measure-iT high-sensitivity nitrite assay kit (ThermoFisher Scientific). Fluorescence readings were recorded at λ_ex/em 365/450 nm.

To quantify the cumulative level of NO produced, the more stable oxidation product nitrite/nitrate was measured using the Measure-IT High-Sensitivity Nitrite Assay Kit (ThermoFisher Scientific) according to the manufacturer’s protocol. Assay samples were prepared in the dark. Briefly, cells were plated at 1 × 10⁶/60-mm plate and maintained overnight. Cells were maintained in 2 ml of the fresh medium containing 5% Matrigel for the designated time periods. The CM was harvested and spun to remove the Matrigel drip. 10 μl of the cleared CM was analyzed for nitrite concentration using nitrite standards at the excitation/emission maxima of 340/410 nm.