Synergy mx multi mode plate reader

Intracellular NO production was measured via the fluorimetric Nitric Oxide Synthase Detection System (Sigma-Aldrich), which is based on the iNOS-dependent conversion of a cell-permeable diacetate derivative of 4,5-diamino-fluorescein (DAF-2 DA) to the membrane- impermeant fluorescent molecule triazolofluorescein (DAF-2T) via a 4,5-diamino-fluorescein (DAF2) intermediate. Fluorescence of DAF-2T was measured using a Synergy MX multimode plate reader (Bio-Tek, Winooski, VT) with an excitation wavelength of 490 nm and an emission wavelength of 520 nm.
RAW264.7 macrophages were seeded into wells of a black-walled, clear bottom 96-well tissue culture plates and incubated for 2 hours to allow for adherence. Macrophages were washed with PBS and stimulated with LPS/IFNγ in either RPMI media or co-culture supernatant for ∼24 hours before initiating the iNOS activity assay according to the manufacturer's protocol.

An aliquot (1 mL) of nylon-6 or nylon-11 NPs was transferred to a tared Eppendorf tube and placed in a vacuum oven (Model AccuTemp-09 k, Across International, Livingston, NJ, USA) overnight, and was weighed the next day. The dried particles were dissolved in formic acid (1 mL, nylon-6) or HFIP (1 mL, nylon-11) and their fluorescence was determined using Synergy MX multi-mode plate reader (BioTek Instruments, Inc., Winooski, VT, USA). Calibration curves of NR and ATRB in formic acid were obtained via serial dilutions of the fluorophore (NR in formic acid: 10 µg/mL stock solution, λ_ex = 590 nm, λ_em = 670 nm; ATRB in formic acid: 10 µg/mL stock solution, λ_ex = 560 nm, λ_em = 590 nm; NR in HFIP: 5 µg/mL stock solution, λ_ex = 610 nm, λ_em = 670 nm; ATRB in HFIP: 62.5 ng/mL stock solution, λ_ex = 550 nm, λ_em = 580 nm).

In-vitro drug release assays involved incubation of the implants in 40 ml of 1X phosphate buffered saline (PBS, pH 7.4 at 37°C) on an orbital shaker at 100 rpm. TAF species in the release media were measured by ultraviolet-visible (UV) spectroscopy at 260 nm using the Synergy MX multi-mode plate reader (BioTek Instruments, Inc., Winooski, VT, United States). The release buffer was sampled twice a week during which time the implants were transferred to 40 ml of fresh buffer to maintain sink conditions. The quantity of TAF species (TAF and tenofovir-containing species) released in the PBS buffer during the time interval was calculated, and the cumulative mass of drug release as a function of time was determined.