Automated Cell Counting and Viability Assay

Cell count was determined by quantifying nuclei per images, following DAPI staining. This task was performed automatically using Lionheart FX automated microscope using GEN5 Microplate Reader and Imager Software Version 3.05 for imaging and quantification, both from BioTek Instruments Inc. (Vermont, USA). Additionally, we determined cell viability via biomass performing a crystal violet assay using a protocol previously described (Del Favero et al. 2021b (link)). Briefly, cells were rinsed with warm DPBS before fixation with cold ethanol (99%). Staining was performed for 5 min using crystal violet solution (0.1%). Cells were washed 4 times using autoclaved water. Cells were lysed for 10 min on an orbital shaker (500U/min) using 99% EtOH and acetic acid (1%), before measuring absorbance [595 nm; Cytation 3 multi-mode plate reader (BioTek Instruments, Winooski, VT, United States)]. Three independent cell preparations were prepared and three technical replicates were measured.