In cell analyzer 2000

Cell lines were seeded at 30,000 cells/well in a 96-well plate and allowed to adhere for 24 h overnight in an incubator at 37 °C under 5% CO₂. Cells were then treated with 100 µL of a 1000 µg/mL Zein:PEG-Zein NP formulation coupled to CF-647, as described in Section 2.3. The variation to the protocol was made to incorporate CF-647 dye at a molar ratio of 1:0.002 Zein to dye by reacting the Zein to the dye in the same manner as the PEG-NHS ester. The formulation was then subjected to a Sephadex G-50 spin column using HBS as the exchange buffer and was sterile filtered. At fixed time points (e.g., 2, 4, 8, and 16 h), media containing the Zein:PEG-Zein NPs were removed, and the cells were washed three times before being stained with Hoechst 33342 (2 µM) for 20 min preceding imaging. Cells were then imaged with the InCell Analyzer 2000 using Bright Field, DAPI, and Cy5 excitation and emission filter.

NPC fibroblasts (I1061T/I1061T, GM18453, obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research) were grown on glass-bottomed 96-well plates (#164588, Nunc) and treated as indicated. Filipin staining for evaluation of cholesterol accumulation was performed as previously described [20 (link)], and images were acquired on an IN Cell Analyzer 2000 using the following settings: 60x (air) objective lens, 2x2 binning, Quad1 dichroic mirror and DAPI/DIC filters, with hardware autofocus, plate heater 30°C, and exposure time for DAPI (filipin) 0.100 sec and bright field 0.100 sec. Nine fields of images separated by 300 μm were acquired for each well. Quantification of relative filipin intensity, defined as filipin intensity within high-intensity lysosome-like organelles per total filipin intensity within cells, was performed using R and the EBImage package [24 (link)]. Details of the image processing are summarized in S1 Fig, and an example R script for the image analysis is available from the Mendeley Data repository (http://dx.doi.org/10.17632/jr23ccpp46.3).

The images of immunofluorescence were acquired using the automated high throughput fluorescent microscope IN Cell Analyzer 2000 with a Å~20 objective. The fluorophores in wavelength settings were used to generate the IF images (‘DAPI’ for DAPI, ‘FITC’ for AlexaFluor 488 FITC, and ‘Cy5′ for AlexaFluor647). Multiple fields per well were acquired to include a minimum of 100 cells per sample well.
High content analysis (HCA) of the images were processed using the INCell Investigator v.2.7.3 software as described previously (Borghesan et al., 2019 (link), Rapisarda et al., 2017 (link)). DAPI was used as a nuclear mask and a top-hat method allowed the segmentation of cells. To detect cytoplasmic staining in cultured cells, a collar of 7–9 nm around DAPI was applied. Nuclear staining in the reference wavelength, that is all the other wavelengths apart from DAPI, was quantified as an average of pixel intensity (gray scale) within the specified nuclear area. Cytoplasmic IF staining was quantified as a coefficient of variance of the pixel intensities within the collar area in the reference wavelength. In samples of cultured cells, a threshold for positive cells was assigned above the average intensity of unstained or negative control samples.

A549 lung adenocarcinoma cells, 293t embryonic cells, Mia PaCa-2 pancreatic carcinoma cells, HDF cells, HT-29 colorectal adenocarcinoma cells, RAW 264.7 macrophage cells, J774A.1 macrophage cells, and MDA-MB-231 breast adenocarcinoma were purchased from ATCC (Manassas, VA, USA). A549 and MDA-MB-231 cell lines were cultured in RPMI 1640 medium, 293t, RAW 264.7, J774A.1, Mia PaCa-2, and HDF cell lines were cultured in DMEM medium, and HT-29 cells were cultured in McCoy 5A medium. All media were supplemented with 10% fetal bovine serum and 2 mM L-glutamine if instructed by the manufacturer. All in vitro experiments were performed between the third and eighteenth passages. All cell lines tested negative for mycoplasma. Cell doubling times were determined by plating 5000 cells/well of each cell line in triplicate for each future time point (24, 48, 72, and 96 h) on a 96-well plate. At the indicated time points, the cells were stained with Hoechst 33342 (2 µM) and ethidium homodimer (2 µM) for 20 min before being imaged using the InCell Analyzer 2000 under the DAPI and Cy3 filter sets.

The cell cycle profile analysis by DNA content measurement was performed using two fluorescent dyes (nuclei: DAPI at final concentration 1 µg/ml; cell cytoplasm: Calcein-AM at final concentration 0.3 µg/ml). Images were captured using InCell Analyzer 2000 and the output was processed by the ImageJ software with the DNA cell cycle plug-in. Cell cycle phases in a given cell population were presented as % of cells in G0/G1, S and G2/M phases (cell cycle distribution) or as % of micronuclei positive cells (micronuclei formation). A minimum of 1000 cells were counted in each sample.

After various treatments, HUVECs were co-incubated with THP-1 cells labeled with Hoechst 33342 for 3 h in the dark at 37°C. The non-adherent THP-1 cells were washed gently with PBS. Images were obtained under random fields of each well using IN Cell Analyzer 2000.