Operetta imaging system

On the 45th day of differentiation, cerebral organoids were fixed in 4% paraformaldehyde, incubated with sucrose solutions (10, 20, and 30%) in phosphate buffered saline (PBS), embedded in optimal cutting temperature compound (OCT), and frozen in liquid nitrogen. The organoids were sectioned with a cryostat into 20 μm thick sections. Immunofluorescence was performed using the primary antibodies: anti-MAP2 (M1406, Sigma-Aldrich), anti-AMPAR1 (Abcam, ab86141), anti-NMDAR1 (Abcam, ab28669), anti-sigma receptor 1 (sc-137075, Santa Cruz), and anti-5-HT_2A receptor (RA24288, Neuromics). Secondary antibodies used were as follows: Alexa Fluor 488 goat anti-mouse (A11001, Invitrogen) and Alexa Fluor 594 goat anti-mouse (A-11008, Invitrogen). DAPI was used for nucleus staining. Images were acquired using an Operetta Imaging System (Perkin Elmer) and a Leica TCS SP8 confocal microscope, when specified.

Day 10 MN progenitors were plated on poly-D-lysine/laminin-coated 96-well plates at a density of 5,000 cells per well and differentiated into neurons. Cells were immunostained at day 30 (Supplemental Experimental Procedures) and images were captured on the Operetta imaging system (PerkinElmer) in an automated fashion. MNs and non-MNs were detected based on ISL1 and TUJ1 staining and quantified by the Columbus software using standard pipelines. Cells were cultured further for 14 days, immunostained, and counted as above. Counts at day 44 were normalized to those obtained at day 30. Motor neuronal soma and neurites were detected by first identifying ISL1+ nuclei and then demarcating soma and neuronal processes using Columbus pipelines. Apoptotic cells at day 37 were detected using the CellEvent Caspase 3/7 green detection reagent (Thermo Fisher) according to the manufacturer's instructions. The reagent was added to live neurons and incubated for 30 min, then counterstained with the nuclear dye Hoechst 33542 (Molecular Probes).

To determine IUs produced during infection, cell culture supernatant from infected cells was collected at 120 or 144 HPI. To determine cell-associated virus titer, cells were washed with PBS and collected by scraping into complete growth media. The cells were lysed by subjecting them to one freeze-thaw cycle and bath sonicating to release cell-associated virus. To generate the virus titer reporter plate, cells were seeded in 96-well dishes. Virus-containing media collected from either cell culture supernatant or cell-associated samples were used to infect the reporter plate in a dilution series (0.1–0.001). After 24 hours, reporter plate cells were washed with PBS and permeabilized with ice-cold methanol at −20°C for 15 min. Following permeabilization, the cells were washed with PBS and blocked with 3% BSA in PBS + 0.2% Tween 20 (PBST) for 1 hour. After blocking, the cells were incubated in 1:40 mouse anti-IE1 (clone 1B12, gift from Thomas Shenk) in 0.3% BSA in PBST for 1 hour. Following primary antibody incubation, the cells were washed with PBST and incubated with 1:1,000 goat anti-mouse AlexaFluor 488 and 1:1,000 DAPI in 0.3% BSA in PBST for 1 hour. The cells were then washed with PBST, and the number of infected cells was quantified via immunofluorescence detection of IE1 using the Operetta imaging system (PerkinElmer).

Biofilm quantification was carried out according to the protocol of O’Toole [6 ]. Absorbance quantified using microtiter plate reader (Spectramax M2, Molecular Devices, USA). Cells were grown on 96-well costar plates and incubated at 37°C for 24 h for visualization of biofilm development. After the incubation, the wells were washed with sterile phosphate-buffered saline and stained with acridine orange (0.1% w/v), and the biofilms were imaged using high-content screening (HCS) using Operetta imaging system (Perkin Elmer, Germany). The Z-stack analysis performed using Harmony software 2.0.1.

SH-SY5Y cells were seeded in 24-well plates and transfected with siCtrl and siALK #1 using Lipofectamine 2000. After 52 h, cells were synchronized with 4 µM RO-3306 for 20 h. After being washed with pre-warmed PBS (+) four times at 37°C on a water bath, pre-warmed DMEM containing 5% FBS and 0.1 µM Hoechst 33342 was added to the cultures. Immediately, the 24-well plate was set in the live cell chamber of an Operetta imaging system (PerkinElmer, Waltham, MA, USA), and live cell images of bright field and fluorescence of Hoechst 33342 were acquired every 3 min for 5 h at 37 °C in 5% CO₂ [54 (link)]. Likewise, SH-SY5Y cells were treated with 1 µM crizotinib or DMSO immediately after RO-3306 release and live cell images were obtained as mentioned above. Similarly, H2228 cells were imaged in the presence of DMSO or 0.7 µM TAE684 using the same Operetta imaging system. Time-lapse images were captured every 5 min for 12 h. The duration of prophase/prometaphase was measured from mitotic entry (chromosomes condensation/cell round up) to metaphase (chromosomes alignment at the cell equator). The time from chromosomes alignment to their separation and from chromosomes separation to cleavage furrow completion were defined as metaphase and anaphase/telophase, respectively.