Evos fl auto 2

HPSC-cardiomyocytes were seeded as full monolayers (30 K cells per well) onto vitronectin-coated CellCarrier-96 well special optics plates (PerkinElmer) and were treated with 1 μM of the anticancer drugs DOXO, AMR or ACLA 10-12 days after seeding. Images of hPSC-cardiomyocytes were acquired using the high-throughput automated EVOS FL Auto 2 (Thermo Fisher) microscope equipped with a 40x Super-apochromat Olympus objective (NA 0.95) (Thermo Fisher, AMEP4754). Anthracyclines were quantified based on auto-fluorescence using the RFP filter cube (ex531/20nm; em593/40nm, Thermo Fisher, AMEP4652). The cell culture area of the well was scanned by automatically acquiring 55 images per well at indicated timepoints. Cells were maintained during the 24 hours at 37°C and 5% CO₂ on the EVOS FL Auto 2 with the EVOS Onstage incubator (Thermo Fisher). For flow cytometry quantification, cardiomyocytes were treated in suspension with the compounds and autofluorescence was quantified (at ex561nm, em586/15) with a MACSQuant VYB flow cytometer (Miltenyi Biotech) at the indicated time points. Plots were analyzed with FlowLogic software.

To visualize the biofilm after treatment with Lycosin-II, we used calcofluor white (10 μg/mL) to stain C. albicans in the biofilm. Moreover, the S. aureus (ATCC 25923) strain expressing GFP allowed for the visualization of this bacterium. The images were visualized using EVOS FL Auto 2 fluorescence (Invitrogen).

Sperm samples from five cats (three replicates) were incubated at 38.5 °C in SOF medium +1000 IU of Penicillin, 10 μg/ml streptomycin, 10 μg/ml of heparin, 20 µM penicillamine, 10 µM hypotaurine, 1 µM epinephrine for up to 24 h and samples collected at 0, 1, 2, 18, and 24 h for motility analysis; or in PBS with or without oviductal EVs and samples collected at 0, 0.5, 1, 2 and 3 h for acrosome integrity. For motility analysis, 10 μl of sperm samples were dropped onto a glass slide, covered with a coverslip and observed at a magnification of 200x on a positive phase-contrast microscope (Leika Leitz DM1L; Wetzlar, Germany) with a warmed (38 °C) stage. Ten fields per sample were recorded using a Nikon DS-F camera (Japan), and a minimum of 100 spermatozoa were analyzed in each sperm sample for sperm total motility (percentage of moving spermatozoa), hyperactive (star spin moving spermatozoa) and progressive (forward moving spermatozoa) motility analyses. For acrosome integrity assessment, 10 μl of sperm were fixed in 10 μl of 4% paraformaldehyde for 15 min and labeled with HOECHST33342 (for DNA) and the acrosome-specific fluorochrome fluorescein isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin (FITC-PNA). At least one hundred spermatozoa were counted at 1,000x magnification (EVOS FL auto 2, Invitrogen, USA),

MDMs were prepared for immunofluorescence staining. 4% formaldehyde and 100% cold methanol were used for fixation and permeation, respectively. Blocking was performed with blocking buffer at RT for 60 min. Cells were then incubated with rabbit anti-HIF-1α (1:500) or anti-β-actin (1:5,000) overnight at 4°C. The immune complexes were glowed using Alexa Fluor 488 goat anti-rabbit (1:1,000) or Alexa Fluor 594 goat anti-mouse (1:1,000) secondary antibodies before counterstaining with DAPI (Solarbio). Images were obtained using a fluorescence microscope (EVOS FL Auto2; Invitrogen), and image processing was performed using ImageJ software.

CMT and LLC cells were plated on Collagen (Rat Tail Type I) or Matrigel and allowed to adhere for 2h at 37°C. Cells were fixed with 4%PFA and nuclei were stained via incubation with DAPI (1:1000) for 10min. Fluorescent images were acquired on Evos FL Auto 2 fluorescent microscope (Invitrogen). Tile-scans were obtained using a 4x air objective with 3.2 MP CMOS camera. Excitation using DAPI LED light cube was used. Images were acquired using EVOS software (v2). Tiles were knitted into.TIFF files using FIJI software and total cell count was obtained by thresholding for nuclear stain followed by automated counting. 35mm low stiffness μ-dishes (1.5kPa and 28 kPa) were obtained from Ibidi.

Adipogenic and osteogenic differentiation were utilized to prove the multilineage of ADSCs. ADSCs at 80–90% confluence was incubated with the specific medium of adipogenic differentiation (OriCell, HUXMD-90031, Cyagen) for two weeks and osteogenic differentiation (OriCell, HUXMD-90021, Cyagen) for three weeks. Subsequently, the paraformaldehyde-prefixed ADSCs were identified with lipid droplet and calcium nodules under an optical microscope through Oil Red O Solution and Alizarin Red S. Images were obtained by Evos FL Auto2 (Invitrogen, Thermo Fisher Scientific).