3d cell explorer fluo microscope

BUVEC were seeded into 35 mm tissue culture μ-dishes (Ibidi^®) and cultured (37°C, 5% CO₂) until confluence. Ezetimibe treatment (20 μm) was performed as described above. Thereafter, T. gondii, N. caninum and B. besnoiti tachyzoites were used to infect cell layers (MOI = 3:1). At 24 h p. i., holotomographic images were obtained by using 3D Cell-Explorer-fluo microscope (Nanolive) equipped with a 60 × magnification (λ = 520 nm, sample exposure 0.2 mW/mm²) and a depth of field of 30 μm. Images were analysed using STEVE software (Nanolive) to obtain refractive index (RI)-based z-stacks (Silva et al., 2019 (link)). Additionally, digital staining was applied according to the RI of intracellular tachyzoites. Finally, intracellular meront development was evaluated by counting intra-meront tachyzoites in at least six 3D holotomographic z-stacks of infected host cells ( = 50 cells per condition) in presence or absence of ezetimibe (20 μm).

Hemocytes (n = 100) were resuspended in imaging medium, which is Grace’s insect medium containing Hoechst (diluted 1:1000) to label DNA and SYTOX Green (diluted 1:2000), to label dead cells. For 3D holotomography, hemocytes in imaging medium were seeded into 35 mm low-rimmed tissue culture µ-dishes (Ibidi^®, Gewerbehof, Germany) and allowed to settle for 10–15 min. Images were acquired using a 3D Cell Explorer-fluo microscope (Nanolive^®, Tolochenaz, Switzerland) equipped with 60× magnification (λ = 520 nm, sample exposure 0.2 mW/mm²) and a depth of field of 30 µm and an Ibidi^® top-stage chamber (Ibidi^®, Gewerbehof, Germany ) to keep the temperature stable (RT). At the end of the experiment, images were analyzed using Steve^® software v.2.6 (Nanolive^®, Tolochenaz, Switzerland) to obtain refractive index (RI)-based z-stacks [37 (link)]. Further, 3D rendering and digital staining were performed based on RI values and thereafter illustrated. Additionally, each channel was exported separately using Steve^® software v.2.6 (Nanolive^®, Tolochenaz, Switzerland) and managed with Image J Fiji v1.7 (NIH, Bethesda, MD, USA) as described elsewhere [38 (link),39 (link)].

A549 cells in complete medium were grown on 12-mm cover glasses in 24-well cell culture plates. The cells were then treated with the prepared CM. After 24 h, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 15 min. After blocking with 1% BSA for 1 h, the cells were stained with Alexa Fluor 594 phalloidin for 40 min at room temperature (15‒25 °C). The cells were then washed twice with PBS and mounted with Vectashield mounting medium containing DAPI (cat. no. H-1500; Vector Laboratories, Burlingame, CA, USA); fluorescence images were acquired using a 3D Cell Explorer Fluo microscope (Nanolive, Ecublens, Switzerland).