Incucyte zoom 2016b analysis software

The apoptotic effect of staurosporine on primary cell cultures (7 × 10⁴ cells/well) was analyzed on day 13 of the cultures. For this endeavor, 1 mM staurosporine (Cell Signaling Technology, Leiden, the Netherlands, #9953) diluted in dimethyl sulfoxide (DMSO) was added to culture medium (1:1000 dilution ratio, final concentration 1 μM dm⁻³) along with Incucyte^® Caspase-3/7 Dye for Apoptosis (Sartorius AG, Göttingen, Germany, #4440) and Incucyte^® Annexin V Dye for Apoptosis (Sartorius AG, #4641) diluted 1:1500 and 1:200, respectively.
The progression of the development and distribution of apoptotic markers in the staurosporine-induced apoptotic cell population was monitored using the IncuCyte™ ZOOM system (Essen BioScience, Ann Arbor, MI, USA) for Caspase-3/7 Dye (excitation [Ex]/emission [Em] 500/530 nm) and for Annexin V Dye (Ex/Em 593/614 nm). Analysis was performed using the IncuCyte ZOOM 2016B analysis software (Essen BioScience, RRID:SCR_019874) on nine microphotographs for each well (n = 4 wells) taken with a ×10 objective lens (Supplemental Table 1) every 30 min for a total of 24 h. For both markers, individual analysis was performed uniformly for each captured microphotograph (Supplemental Table 2). The results were compared with control data obtained in a similar manner from a primary cell culture that was cultivated simultaneously in staurosporine-free medium.

Immunofluorescent co-localization using anti-tubulin β 3 (clone TUJ1) and anti-APC (clone CC1) antibodies was realized to analyze the presence of cC3 and cPARP in neuronal and oligodendroglial populations of primary cell culture treated with staurosporine. Uniform analysis procedure (Supplemental Table 2) was prepared with the IncuCyte ZOOM 2016B analysis software (Essen BioScience) and used to estimate the number of cC3⁺ and cPARP⁺ cells per mm² in 14 microphotographs taken with a ×10 objective lens from each well (n = 6 wells per experimental group). Co-localization of apoptotic (cC3, cPARP) and phenotypic (TUJ1, CC1) markers in cells was identified as signal overlap and performed on three wells per experimental group. Co-localization was evaluated as percentage of neurons or oligodendroglial cells.