Operetta cls high content analysis system

1,500 transfected cells (24 h after transfection) were seeded in 384-well plate (781090; Greiner Bio-One) and incubated overnight in normal growth conditions. Cells were starved in low serum condition (1% FBS) for 24 h and treated with 20 ng/ml HGF for 10- or 30-min. Cells were fixed directly with 4% PFA at 37°C for 20 min. Between all subsequent steps, cells were washed five times with PBS using the 50 TS washer (BioTek). Cells were permeabilized with 0.05% saponin for 20 min at RT. Blocking was performed during 1 h at RT with 1% FBS. Primary antibody (Table S7) solution was added to the sample and shaken at 100 rpm on a HS 500 horizontal reciprocating shaker (Janke & Kunkel) for 2 h. Secondary antibody (Table S7) solutions were added to the sample and shaken at 100 rpm for 2 h. Nucleic acids were stained with Hoechst-33342 (B2883, 1:2,000 in PBS; Sigma-Aldrich) for 20 min at RT. Image acquisition was performed using the Operetta CLS High-Content Analysis System (HH16000000; PerkinElmer) at 40× magnification. Nine fields per well with 9 z-planes with 500 nm z-spacing were acquired per site. Maximum intensity projection was computed and used for subsequent analysis. For puncta quantification, EEA1 or LAMP1 signals were segmented using the Harmony software (PerkinElmer) and quantified. The total number of puncta was referred to the number of cells present per field.

The cells were fixed in 4% PFA-PBS (Thermo Scientific, Waltham, MA, USA) for 15 min and permeabilized with 0.1% Triton-X-PBS (Fisher Scientific, USA) solution for 10 min at room temperature. Blocking was carried out with 3% BSA-PBS (Lee Biosolutions, Maryland Heights, MO, USA) for 1 h. Viral infection was detected using primary antibody grown in rabbit against CHIKV nonstructural protein 3 (nsP3) (1:1000; Dr. Andres Merits) or mouse anti-E1 (viral structural protein) (1:500; BEI Resources, Manassas, VA, USA) incubated overnight at 4 °C. Cells were then stained with secondary antibody, Alexa 488-conjugated goat anti-rabbit (1:2000, ThermoFisher Scientific, Waltham, MA, USA), for 1 h at room temperature. Cell nuclei and cytoplasm were labeled with Hoechst 33342 and the CellMask Deep Red Plasma membrane stain (Invitrogen), respectively. High-content quantitative imaging data were acquired on an Operetta CLS High Content Analysis System (PerkinElmer, Waltham MA, USA). Image analysis was accomplished using Harmony High Content Analysis software. Percentage inhibition was calculated using mean percentage infection from the infected and untreated cells as a positive control (100% inhibition).

Fluorescence images of whole brain sections were acquired using a 10 × objective lens on the Operetta® CLS™ High Content Analysis System (PerkinElmer). The cortex and hippocampus from 3–4 brain sections per mouse were manually segmented and the area covered by Aβ42-positive staining, as well as thioflavin S-positive fibrillar dense-core plaques, was quantified using the Fiji software by applying an automated local threshold that was maintained for all images analyzed. For each mouse, the total cortical and hippocampal area (%) covered was determined by calculating the average of all captured sections.

A previously described immunofluorescence protocol was followed [7 (link)]. Briefly, 48 h after the hESCs were transduced with shSc and shPUM1-2, the cells were fixed with 4% paraformaldehyde, rinsed with PBS, and incubated with blocking buffer (PSA/BSA 5%) for 60 min. The cells were subsequently incubated for 60 min at 30°C with primary antibodies for PUM1 (1:300, Bethyl Laboratories Inc.), PUM2 (1:70, Bethyl Laboratories Inc.) or OCT4 (1:100, Abcam), which were diluted in blocking buffer. After three washes with PBS, the cells were incubated with an Alexa Fluor® 488 anti-goat or anti-rabbit secondary antibody for 60 min at 30°C. DAPI staining was performed for 10 min, and the cells were then washed three times with PBS. Images were acquired at 20x magnification using an Operetta CLS High-Content Analysis System (PerkinElmer), and the staining intensity of each individual cell (n = 74,000) was analyzed using Harmony 4.5 software (PerkinElmer) (S2 Fig). The experimental design and control images of the cells incubated with secondary antibody are detailed in S3 Fig (S3A–S3B Fig).

The procedures for assessing cell viability and plate colony formation were described previously [18 (link), 19 (link)]. The cumulative motility of CC cells was calculated using an Operetta CLS™ high-content analysis system equipped with Harmony software (Perkin Elmer, Waltham, MA, USA). The measurement was performed in digital phase contrast mode at 37 °C and 5% CO₂ using a × 20 long distance objective. In addition, apoptosis and cell cycle assays and western blots (WBs) were performed [18 (link)].

Colonic tumor tissues were received from consented patients following Institutional Review Board (IRB) approval at the Norris Comprehensive Cancer Center of the University of Southern California, Los Angeles, CA, USA. Colonic tumor specimens were isolated from colon cancer patients and processed mechanically and enzymatically to obtain colon cancer cells without contaminating non-cancer cells as previously described^{37 (link)}. Patient-derived organoids (PDOs) were generated from these colon cancer cells as described in our recent publication^{6 (link)} and treated with B32B3 and Taz individually or in combination for 5 days. At the end of treatment, 100 μl of CellTiter-Glo 3D solution (Promega, WI, USA) was added to each well, and cell viability was analyzed by measuring luminescence with BioTek Synergy Neo2 Multi-Mode Reader (Agilent Technologies, CA, USA). Images of PDOs were also acquired 0 and 5 days after inhibitor treatments using the Operetta CLS high-content analysis system (PerkinElmer, MA, USA) and displayed with maximum intensity projections of 24 z-stacks ranging from 10–470 µm in increments of 20 µm.