Cytell cell imaging system

The cell lines were seeded at a density of 10,000 cells/well in 48-well plates (Thermo Scientific Nunclon^TM Delta Surface-150687). During immunocytochemistry, the growth medium was discarded, and cells were washed thrice gently with Dulbecco’s modified PBS (DPBS; pH 7.5). Briefly, cells were fixed in 4% paraformaldehyde ± 0.1% Triton X-100 (depending on the desired permeabilization conditions), rinsed with DPBS, and incubated with primary antibodies at 4 °C overnight. After rinsing again in DPBS, cells were incubated with an appropriate fluorescence-conjugated secondary antibody (Supplementary Table S2) and with diamidino phenyl indole (DAPI) as a nuclear stain (diluted to a final concentration of 1 µg/mL) for 2 h at room temperature in the dark with gentle rotary shaking. The plates were then washed thrice with DPBS and images were captured on a high-content imaging platform (Cytell Cell Imaging System (GE Healthcare) or IN Cell Analyser 6000 (GE Healthcare, Buckinghamshire, UK), as indicated), with approximately 6–9 fields of view taken per well. Images were further analysed and quantified using the IN Cell Investigator software v1.0 (GE Healthcare).

Cells were seeded into 6-well plates and allowed to grow for 48 h before treatment. The cells were fixed, permeabilized and stained for γ-H2AX or Rad51 by using specific antibodies. Plates were imaged with a × 10 objective using a Cytell Cell Imaging System (GE Healthcare, Chicago, Illinois, USA). MyBioApp Protocol with specified parameters was created to acquire the data and to quantify the signal intensity in nuclei, cytoplasm, and in whole cells. DAPI (blue channel) was used for the nuclear masks, whereas Alexa Fluor 647 Mouse (red channel) was used for γ-H2AX-staining and Alexa Fluor 647 Rabbit (red channel) for Rad51-staining. Graphics illustrating the average intensity of the nuclear γ-H2AX-staining and Rad51-staining were automatically generated by using MyBioApp Protocol and then passed to MS Excel for further processing and analysis.

For this analysis, 3T3 cells were seeded in 96-well plates at a density of 3 × 10³ cells/well. The culture medium was added to 400 µL for each well. Wells were covered with parafilm tape. The 3T3 cells underwent SMG for 72 h. The control group was under 1G in the same CO₂ incubator. After 72 h, the culture medium was discarded. The nuclei of 3T3 cells were stained with Hoechst 33342 (14533, Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The 3T3 cells were washed 3 times with phosphate-buffered saline (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were observed under fluorescence with a Cytell cell imaging system (GE Healthcare, Chicago, IL, USA) and cell number was determined by counting nuclei with the Cytell’s cell cycle app. The cell cycle app was also used to evaluate cell cycle progression and nuclear morphology parameters, including nuclear intensity, shape, and area. In order to assess the nuclear properties of the 3T3 cells, the parameter of nuclear area (µm²) was adjusted to 150 and the parameter of sensitivity (%) was adjusted to 50 (according to the Cytell manual).

The evaluation of the toxicity of the IA-12 and IA-12/oNu systems was estimated by means of the multifunctional Cytell Cell Imaging system (GE Health Care Life Science, Sweden) using the Cell Viability Bio App. Cell lines M-HeLa (epithelial carcinoma of the cervix, HeLa subline, M-HeLa clone) were acquired from the Type Culture Collection of the Institute of Cytology (Russian Academy of Sciences, Saint Petersburg, Russia) and Chang liver normal cells from the N.F. Gamaleya Research Center of Epidemiology and Microbiology (Moscow, Russia). The cells were seeded in a concentration of 10⁵ cells/mL in 96-well plates (Eppendorf, Hamburg, Germany), then 150 μL of standard Eagle’s medium (PanEco, Moscow, Russia) was added per well, and incubation proceeded with CO₂ at 37 °C. The medium was then supplemented with 10% fetal calf serum and 1% nonessential amino acids. Twenty-four hours after seeding, the systems under study were added at preset dilutions, 150 μL to each well. The dilutions were prepared immediately in a nutrient medium. The experiments were repeated three times. Intact cells cultured in parallel with experimental cells were used as a control.

At 78 or 72 h post-transfection, the nuclei of cultured cells were blue-stained by Hoechst 33342 live cell stain and viewed under Cytell cell imaging system (GE Healthcare Life Science, Buckinghamshire, UK) to detect the expression of Cas9-RFP. Successful transfection yields red fluorescence color. Blue and red channels photographs of random fields of HepG2 cells were taken at high magnification (field of view 880 × 660 µm). All imaging parameters, like exposure time and gain, were fixed for all photographs to obtain reliable images to be used for quantification. Three photographs for each channel were analyzed by two independent observers using ImageJ software (NIH, Bethesda, MD, USA). The transfection efficiency was calculated by dividing the number of RFP-positive cells by the total number of surviving cells in the photographs. [97 (link)]. In addition, the kinetic of gene expression was monitored for 48 and 72 h, and it was evaluated by quantifying the mean red florescence intensity using ImageJ software [95 (link)].

FTD was detected as described in our previous report^{13 (link)}. Briefly, cell seeding and addition of doxycycline and FTD were performed as described for the cell viability assay. One hour after FTD addition, cells were fixed in ice-cold 70% ethanol for 5 min and treated with 1.5N HCl for 1 h. After blocking with goat serum, FTD was detected with an anti-BrdU antibody (1:250; clone 3D4; BD Biosciences). Samples were treated with an Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1:200; Thermo Fisher Scientific) and 1 μg/ml 4′, 6-diamidino-2-phenylindole, dihydrochloride (DOJINDO). Fluorescence was detected with a Cytell Cell Imaging System (GE Healthcare). Images were analysed using IN Cell Analyzer Workstation 3.7.1. The maximum fluorescence intensity in cells cultured without FTD was determined as the background. Outliers and signals less than the background were excluded. To calculate outliers, the upper quartile (Q_3/4) and lower quartile (Q_1/4) were first determined. Then, the interquartile range (IQR) was calculated as Q_3/4–Q_1/4. Data that were more than Q_3/4 + 1.5 × IQR or less than Q_1/4 − 1.5 × IQR were defined as outliers.

MCF-7 cells were grown for 24 h at 37°C in RPMI-1640 medium supplemented with PoPE (5 μg/mL). The cell cycle was examined by the Cytell™ cell imaging system after staining with the Cytell™ Cell Cycle Kit (GE Healthcare, Tokyo, Japan).