Cytation 3mv cell analyzer

The spheroid cell viability was monitored after 3, 4, 7 and 14 days of culture (D3, D4, D7 and D14) using Live/Dead^® fluorescent microscopy kit (Invitrogen, Carlsbad, CA, USA, catalogue #L3224). Spheroids were incubated for 30 min at room temperature in a working solution containing 1 µM calcein-AM and 2 µM ethd-D1 in D-PBS (Sigma, Saint-louis, MI, USA, catalogue #D8537). They were then transferred to a µ-Slide 8 Well (Ibidi, catalogue #µ-Slide 8 Well) to be imaged using the fluorescent microscopy module of the Cytation™3MV cell analyzer (Biotek^®-M = 4X-fluorescence filters = green fluorescent protein and propidium iodide).

Each spheroid of the microplates was automatically imaged using bright field microscopy module of the Cytation™3MV cell analyzer (Biotek^®-M = 4X) coupled with Gen5 3.08 software (Biotek^®, Winooski, VT, USA). The “Cellular analysis” tool was applied to a stitched picture to calculate the object size of each well (threshold = 20,000, background = light, min. object size = 150 µm, max. object size = 1000 µm). Spheroid size was recorded for each condition on the 1st, 3rd, 4th, 7th, 10th, and 14th days of culture (D1, D3, D4, D7, D10 and D14) allowing growth kinetic curves to be set. Growth curve slopes were calculated for each spheroid condition between the 4th and the 14th culture days (corresponding to the poststorage period) [26 (link)].

For spheroid global hypoxia level evaluation, ROS-ID^® orange reagent (EnzoLifeSciences, Farmingdale, NY, USA, catalogue #ENZ-51042) was used. This probe was attached to a nitro group that was specifically reduced by nitro reductase activity in hypoxic cells, releasing an orange fluorescence. Spheroids were exposed with Hypoxia Red reagent at a concentration of 250 µM diluted in OptiPASS^® medium for 4 h in humid incubator at 37 °C and 5% CO₂ (Figure S1). They were then transferred to a µ-Slide 8 Well (Ibidi, Gräfelfing, Germany, catalogue #µ-Slide 8 Well) to be imaged using the fluorescent microscopy module of the Cytation™3MV cell analyzer (Biotek^®, Winooski, VT, USA, M = 4X, fluorescence filter = Texas Red). Mean fluorescence intensity of spheroids was calculated with “cellular analysis” algorithm of Gen5 3.08 software (threshold = 9000, background = dark, min. object size = 100 µm, max. object size = 1000 µm).