Cytation 5 cell imager

IHC and IF studies using ASCL1 (clone 24B72D11.1, catalog number 556604, BD Biosciences, San Jose, CA) and NEUROD1 (clone EPR17084, catalog number ab205300, Abcam, Cambridge, MA) specific antibodies were carried out on archival formalin-fixed paraffin-embedded tissues. In brief, 5 μm paraffin sections were de-waxed and rehydrated following standard protocols. Antigen retrieval consisted of steaming for 40 min in Target Retrieval Solution (S1700, Agilent, Santa Clara, CA). Slides were then washed and equilibrated in TBS-Tween buffer (Sigma, St. Louis, MO) for 10 min. Primary antibodies were applied at a dilution of 1:25 at 37 °C for 60 min. For chromogenic studies, immunocomplexes were visualized by applying secondary detection reagents of the UltraVision™ Quanto Detection System (catalog number TL-060-QHD, Thermo Fisher, Waltham, MA) following the manufacturer’s instructions. Sequential dual-IF labeling studies were carried out using Tyramide SuperBoost kits (Thermo Fisher, Waltham, MA). All bright-field slides were imaged using a Ventana DP200 system (Roche Diagnostics, Indianapolis, IN). Fluorescence images were acquired on a Cytation 5 Cell Imager (Biotek, Winooski, VT). All the slides have been evaluated by an expert pathologist and the stainings have been replicated a minimum of three times.

Cells were seeded in triplicate in six-well plates under standard conditions until 90–100% confluence was reached, followed by wounding the monolayer by scratching with a micropipette tip, then cells were treated with H₂O₂ 1.0 mM, IC₅₀ (55.24 μM) and IC₅₀/2 (27.62 μM) of 7-hydroxy-3,4-dihydrocalene compound in RPMI medium supplemented with 1% FBS. Wound areas were monitored at four different times (0, 24, 48, and 72 h) using the Cytation 5 cell imager (BioTek Instruments, Inc., Winooski, VT, USA). To determine the migration rate of the cells, the wound areas were quantified using the ImageJ software, (Schneider, Rasband & Eliceiri, 2012 (link)). The variation between the percentage of cell migration (as a function of time) was calculated using the following formula:
$$\mathrm{\%}\mathrm{M}\mathrm{i}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=100-[(\mathrm{A}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{j}\mathrm{u}\mathrm{r}\mathrm{y}\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{t}/\mathrm{A}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{j}\mathrm{u}\mathrm{r}\mathrm{y}\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{z}\mathrm{e}\mathrm{r}\mathrm{o})(100)]$$