Cytocentrifuge

Cells were grown for 48h in standard medium supplemented with 15, 30 or 60nM 6-TG and then irradiated with UVA. Cytochalasin B (Sigma), 4.5 μg/ml, was added in the medium after irradiation, for 24h, then cells were collected and spun onto microscope slides using a cytocentrifuge (Thermo Scientific). Smears were air-dried, fixed 10 minutes in methanol and stained in 4% Giemsa phosphate buffer. Cells were analyzed in the comprehensive micronucleus test as in (30). The frequencies of binucleated cells with MN and NPBs were determined analyzing 500 binucleate cells with a wellpreserved cytoplasm from two slides. The nuclear division index, a cell proliferation index, was determined in 500 cells: [mononucleated cells+(binucleated cells x2)+(trinucleated cells x3)+(tetranucleated cells x4)]/500. Apoptotic cells, having 4 or more than 4 MN, were considered in the analysis.

Embryonic and adult (MI and sham-operated) hearts were fixed in 0.2% paraformaldehyde (Merck) overnight at 4°C, dehydrated in a sucrose gradient (4% followed of 15%), embedded in gelatin, and frozen. Tissue cryo-sections (4 μm thick) were blocked with either 4% FBS-1% BSA blocking solution or Vector M.O.M. basic kit (Vector Laboratories), depending on the specific conditions detailed in S2 Table. Tissue sections were incubated with primary antibodies overnight at 4°C, followed by 1-hour incubation with Alexa Fluor-conjugated secondary antibodies (see S2 Table for the antibodies list; Invitrogen). Slides were mounted, and nuclei were counterstained with aqueous mounting medium with DAPI (Vector Laboratories). Representative high-resolution images were acquired for each heart structure (At, GV-AVJ, and Vt) at 40× magnification in a confocal microscope (Leica SP5II, Leica, Germany). Whole-heart acquisitions were obtained using the high-content imaging system (IN Cell Analyzer 2000, GE Healthcare).
Isolated fixed CMs were resuspended in 10% FBS-PBS and spun onto superfrost slides in a cytocentrifuge (ThermoFisher). Cytospins were incubated with primary antibodies overnight at 4°C, followed by 1-hour incubation with Alexa Fluor-conjugated secondary antibodies. Acquired images were edited and quantified using the Image J version 1.51d software (NIH).

To assess bronchoalveolar lavage fluid (BALF), animals were anesthetized with ketamine (100 mg/kg, Sespo Industry and Commerce, Paulínia, SP, Brazil) and xylazine (10 mg/kg, Vetecia Laboratory of Veterinary Products, Jacareí, SP, Brazil). Then, the trachea was exposed and cannulated with Angiocath with subsequent injection of 4 mL of PBS. Next, samples were centrifuged at 450× g for 5 min. The supernatant was stored at −20 °C for cytokine measurement. Cells were resuspended in 500 μL of RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA), centrifuged at cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, EUA) at 18× g for 3 min, and stained with rapid panoptic (Labor Clin, Sao José do Rio Preto, SP, Brazil) for differential cell count.

Immediately after invasive measurement of pulmonary function, the ends of polyethylene catheters (OD = 0.97 mm Becton Dickinson, Sparks, MD) were secured into the tracheas. Lungs were lavaged 3x with 2.5 ml of Ca²⁺-free and Mg²⁺-free Hanks’ balanced salt solution (HBSS), (Lonza, Rockville, MD), that contained 0.02% EDTA and EGTA. The BAL fluid was centrifuged at 1,000 rpm for 5 minutes at 4°C and the cell pellets were resuspended in 0.5 ml cold HBSS with 10% newborn bovine serum and 0.2% NaN₃. Cells were counted with a Coulter counter (Beckman Coulter, Brea, CA) and 2 x 10⁵ cells were centrifuged onto a glass microscopic slide using a cytocentrifuge (Thermo Scientific, Rockford, IL). Slides were stained with Giemsa stain (Sigma, St. Louis, MO), and at least 200 hematopoietic cells were counted examined microscopically. Cells were characterized as neutrophils, lymphocytes, macrophages, or eosinophils.

A total of 1×10⁵ cells were resuspended to 1mL with PBS. Cells were centrifuged onto glass slides using Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA) at 800rpm for 5 minutes, and then fixed with 4% Paraformaldehyde (PFA) for 15 minutes. Samples were incubated with 0.5% Triton X-100 for 15 minutes at room temperature and blocked with 2% bovine serum albumin (BSA) for 1 hour. The cells were stained with primary antibodies overnight at 4°C, followed by incubation of fluorescent dye-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA). Cells nuclei were stained with DAPI (Sigma Aldrich, MO, USA). Confocal images were obtained with a NIKON confocal microscope.

BALF was collected through lung flushing with HBSS buffer containing 2% bovine serum albumin three times by using a trachea cannula (Angiocath, BD Biosciences). BALF cells were then collected through centrifugation at 300× g for 5 min and spanned on slides by using a cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, USA) and then subjected to Liu’s staining. Monocyte, lymphocyte, neutrophil, and eosinophil numbers within the cell pellet were then counted with a 100× objective lens. The data are presented as the average cell count in at least five fields for each sample. BALF eotaxin and IL-5 levels were determined by using commercial ELISA kits (BD Biosciences).