Fluoroskan ascent microplate fluorometer

SIRT1 deacetylase activity was evaluated in crude nuclear extract from heart samples [5 (link)]. Trichostain A (0.2 mM; Sigma-Aldrich, St. Louis, MO, USA), components of Fluor de Lys SIRT1 Fluorescent Activity Assay/Drug Discovery Kit (Enzo Life Sciences, Farmingdale, NY, USA), including 100 μmol/L fluorogenic peptide encompassing residues 379 to 382 of p53 with lysine 382 being acetylated, and 170 μmol/L NAD+ at 37°C for 1 h, followed by incubation in developer for 15 min at room temperature according to the manufacturer's instructions. Fluorescent intensity was measured using a Fluoroskan Ascent^® microplate fluorometer (Thermo Electron Corp., Milford, MA, USA). No-enzyme and time 0 negative controls were generated by incubating developer II solution with 2 mmol/L NAM before mixing the substrates with or without samples. SIRT1 activity was calculated with the corrected arbitrary fluorescence units of the tested samples to noenzyme control and expressed as fluorescent units relative to the control. The nuclear-cytoplasmic fraction of heart tissue was conducted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo, Fisher Scientific, Rockford, IL, USA) [26 (link)].

To track the uptake of peptides in a time-dependent manner, approximately 0.05 × 10⁶ A673 and EWS502 cells were seeded per well in a 24-well plate and cultured using complete growth medium. 24 Hours post seeding, the cells were washed in serum free medium twice and incubated with 10 μM concentration of FITC labeled peptides (TAT/NLS, TAT/NLS/EWS-PEP) for various time points (1 min, 5 min, 15 min, 30 min, 1Hour, 6Hours and 24 Hours). Following the incubation period, the cells were lysed in Tris buffer (20 mM Tris pH 8, 137 mM NaCl, 1% NP-40 and 2 mM EDTA) and centrifuged. The fluorescence intensity of the supernatant was measured using Fluoroskan Ascent^TM Microplate Fluorometer (Thermo Scientific, Massachusetts, USA) was normalized to the protein concentration.

Both A673 and EWS502 cells were grown in a 100 mm dish for 24 hours and treated with FITC peptides for 1 hour in serum free medium. Preparation of nuclear extract was carried out using NE-PER nuclear and cytoplasmic extraction kit according to the manufacturer’s protocol (Thermo Scientific, USA). The fluorescence intensity was measured using Fluoroskan Ascent^TM Microplate Fluorometer (Thermo Scientific, USA).

FACS analysis was performed to assess the efficiency of the peptide uptake in A673 and EWS502 cells. After incubation with FITC labelled peptides, cells were washed in phosphate-buffered saline (PBS) twice and analysed in Mo-Flo™ XDP (Beckman coulter, USA). A673 and EWS502 cells were washed in serum-free medium and incubated with FITC labelled peptides for 1 hour at 37 °C, fixed in 3% formaldehyde and stained with 0.1% DAPI followed by imaging (Sigma-Aldrich, USA). For measuring intracellular peptide uptake, A673 cells were lysed and the fluorescence was quantified using a Fluoroskan Ascent^TM microplate Fluorometer (Thermo Scientific).

iCell^® neurons and astrocytes were seeded in a 1:1 ratio in the OrganoPlate^® and cultured for 6 days after which the cells were exposed to a concentration range of neurotoxic compounds for 24 hours. For methylmercury and endosulfan the vehicle control was 0.3% v/v DMSO and for 2,5-hexanedione the vehicle control was medium. After 24 hours, the cell viability was assessed using the bio-luminescent RealTime-Glo^TM MT assay (Promega, Madison, WI, USA) according to manufacturer’s instructions. The luminescent signal was measured every 5 minutes using the Fluoroskan Ascent^TM Microplate Fluorometer (Thermo Scientific). For each chip, the medium inlet, the observation window, and the medium outlet were measured and the average value was determined. Three chips were used per condition and the normalized average and standard deviation of each condition were plotted using GraphPad Prism 6 (Fig. 5e).

This assay was an adapted model^{5 (link)} of the method previously published by Gaillard and de Boer⁴⁹ . Before performing the assay, TEER was measured in all transwells (TEER > 100 Ω·cm²). Peptides were prepared at a concentration of 200 μM in Ringer-HEPES buffer containing 20 μM Lucifer yellow (LY) lithium salt (Sigma-Aldrich) as control (P_app < 17·10⁻⁶ cm/s). The apical compartment was filled with 200 μL of the solution containing the peptide, and 800 µL of Ringer-HEPES was poured into the basal well. Three replicates of each peptide were assayed. The plate was left for 2 h in the incubator at 37 °C. Finally, the samples were collected and analyzed or frozen until analysis. LY fluorescence was measured in a 96-well plate with a Fluoroskan Ascent Microplate Fluorometer (Thermo Fisher Scientific). Two parameters were determined, namely transport (T (%)) and apparent permeability (P_app(cm/s)). RP-HPLC-PDA (220 nm) and MALDI-TOF MS were used to determine the amounts of peptide in each sample.