Celldiscoverer 7

9–12-wk gestation placental explants were washed and cut as detailed above. The tissue was then immersed in 1% low melting point agarose (Sigma-Aldrich) at 40°C for less than 30 s, placed in a 48-well plate, and overlaid with 5 μl of additional 1% low melting point agarose. Agarose was allowed to cool for 15 min at room temperature, then IMDM + 10% FCS + penicillin–streptomycin was added to each well and explants were incubated overnight at 5% CO₂ 37°C in a cell culture incubator. Treatments were then added as above, and plates were moved to a Zeiss Cell Discoverer 7 set to an atmosphere of 5% CO₂ 37°C. Oblique brightfield images were acquired with a 2-μm z-step every 30 min for 24 h with an Axiocam 712 mono camera using the Zen imaging software (version 3.4) and a Zeiss Plan-Apochromat 20x/0.7 autocorr lens. Movies and still images were processed in Zen imaging software.

Differentiated IECs were colonized with lactobacilli or not and infected with C. albicans or not (see Table S1). Supernatants were collected, placed in a new 24-well plate and stained for caspase 3/7 activity (CellEvent™ Caspase 3/7 detection reagent; Invitrogen), subsequently the entire wells were imaged at an excitation maximum of 488 nm at 10× magnification in a Cell Discoverer 7 (Carl Zeiss). Images were processed using the Fiji software (ImageJ) and cells were quantified using the Particle Analyser tool.

Sorted primary fibroblasts (PDGFRα⁺EPCAM^-CD31^-CD45^-CD146^-LYVE1^-) from mouse lungs were cultured^{67 (link)}. For single culture in 96-well plates, 2000 cells in 200 μL of a basic medium containing 2% FBS with or without specific drugs, growth factors, or chemokines were used in each well. For co-culture with mouse/human alveolar organoids or empty gel in 24-well plates, 15,000 cells in 350 μL basic medium or equal dose of organoid culture supernatant (both with 2% FBS) were used in each well. Organoid supernatant was collected after centrifugation (1000 g, 10 min). On day 3 of (co-)culture, DsRed fluorescence of the (myo)fibroblasts was imaged with/without staining of Hoechst 33342 (Dojindo). For the live imaging, CellDiscoverer 7 (Carl Zeiss) was used.

Live-cell imaging of the cell death dynamics using PI staining was performed as described previously (78 (link)). In brief, a confluent monolayer of HEK293A cells in a 24-well plate was washed once with culture medium and subsequently infected with 1 × 10⁵C. albicans yeast cells in culture medium or culture medium containing human albumin (Albuman; Sanquin Plasma Products B.V.) concentrations between 10 mg/ml and 0.1 mg/ml. All media contained 4 μg/ml propidium iodide (Sigma-Aldrich). Cells were imaged in a Zeiss Celldiscoverer 7 for 18 h at 37°C and 5% CO₂. Microscopy pictures were taken every 20 min at 10 × magnification from four independent positions per well in the bright field, as well as fluorescence with excitation at 353 nm, and emission was recorded at a wavelength of 465 nm.
Microscopy pictures from the red fluorescence channel were analyzed using Fiji (79 (link)). Using the threshold function, images were converted to binary images, and the number of PI-positive nuclei was enumerated for each image using macro batch analysis and the Particle Analyzer tool. Based on the obtained counts of PI-positive kidney cells, the percentage of dead kidney cells was calculated in relation to the maximal number of dead cells.

EU incorporation assays were performed with a Click-iT RNA Alexa Fluor 594 Imaging Kit (Invitrogen, Cat# C10330). Following treatments, cells were labeled with 1 mM EU for 1 h or 23 h and were then fixed with 4% PFA in PBS for 15 min. Fixed cells were permeabilized with 0.5% Triton X-100 for 15 min. The Click-iT reaction was performed according to the manufacturer’s instructions. Nuclei were stained with 1 µg/ml DAPI, and slides were mounted with VECTASHIELD (Vector Laboratories, Cat# H-1000). Images were acquired with a fluorescence microscope (Celldiscoverer 7, Zeiss) for intensity analysis (Zen v2.6, Zeiss).