Scanr analysis software

10.000 cells were plated per well of a 96 well optical plate. After 24 hours, cells were incubated with 10 μM EdU (Life Technologies, A10044) for 30 minutes, before fixation in 4% PFA. Cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min and washed three times with PBS. Click-iT reaction cocktail containing CuSO₄, L-ascorbic acid and Alexa Fluor 647 fluorescent dye azide in PBS was added to each well and incubated according to the manufacturer’s protocol (Invitrogen C10269). Nuclei were counterstained with DAPI, and cell cycle profiles were made by image acquisition on an automated Olympus IX83 ScanR microscope and Olympus ScanR analysis software.

Wells (6 mm in diameter) of 8-well multitest slides (MP Biomedicals) were coated with fibronectin (Sigma Aldrich) and Cell-Tak (BD Biosciences) as described [18] (link). IgE-sensitized BMMCs were seeded on fibronectin/Cell-Tak-coated wells and allowed to attach for 1 hour. The attached cells were activated for 30 minutes at 37°C with Ag and/or SCF in BSS-0.1% BSA, fixed with 3% paraformaldehyde in glutamate buffer-EGTA (GBE; 137 mM K-glutamate, 2 mM MgCl₂, 3 mM EGTA, and 20 mM PIPES-NaOH, pH 6.8) supplemented with 4% polyethylene glycol, molecular weight 3200, and then permeabilized and stained by one-step exposure to lysophosphatidylcholine (200 µg/ml) and 100x diluted Alexa Fluor 488-phalloidin in GBE. Coverslips were mounted in glycerol-based mounting medium containing Mowiol 4-88 reagent (Calbiochem AG) and Hoechst 33258 (3 µg/ml; Sigma). Fluorescent images (25 images/well) were automatically collected using Olympus IX70 inverted microscope (objective LUCPLFLN Ph1 20x) equipped with motorized stage and ScanR acquisition software (Olympus). Cell area was analyzed using ScanR analysis software (Olympus). At least 500 cells were evaluated in each test.

Quantitative image-based cytometry (QIBC) of the immunofluorescence-stained samples was performed using an automatic inverted fluorescence microscope BX71 (Olympus) using ScanR acquisition software (Olympus) and analyzed with ScanR analysis software (Olympus).

FG-labeled retinas were dissected, fixed with 4% PFA, and incubated twice with PBS/0.1% Triton X-100 for 30 min each. DNA was then stained for 1 min with 50 µg/ml propidium iodide (PI; Sigma) in PBS. Then, retinas were washed five times with PBS, and flat-mounted in glycerol/PBS (1∶1). The relative DNA content of the FG-labeled RGCs was determined as a measure of the integral PI fluorescence values (i.e. integration of total pixel intensity assigned to the visualized nuclei) following a method previously described [12] (link). This analysis was performed using the automated Olympus IX81 microscope-based imaging platform (Olympus), equipped with a digital camera (ORCA-AG C8484-05G01, Hamamatsu Photonics), using 20x magnification objectives. A minimum of 25 fields were acquired in each retina. Analysis of acquired images was performed using Scan^R Analysis software (Olympus). Intensity modules were used for setting the main object “cell soma” (FG staining) and the secondary object “cell nucleus” (PI staining). Basically, cytometry-orientated data analysis was used, setting a first gate of single cells, excluding small particles (less than 30 µm²) or doublets with a dot-plot of area versus circularity. Then, a histogram of PI intensity from the gate of single cells was obtained.