Shandon cytospin 3 cytocentrifuge

Cells were seeded (1000 μL/well) in 24-multiwell culture plates (Orange Scientific, Belgium) at a density of 0.5 × 10⁵ cells/mL and incubated for 24 h under standard cell culture conditions. Cells were then exposed for 72 h to the most promising combination selected in Phase 2 and respective isolated compounds. Briefly, both adherent and non-adherent cells were collected, washed, centrifuged and fixated with 4% (w/v) paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA) in PBS. Then, cells were placed onto silane adhesive microscope slides (VWR International B.V, Amsterdam, The Netherlands) by cytocentrifugation using Shandon Cytospin 3 cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, USA) at 28× g for 5 min. Slides were incubated with DAPI staining solution (1 μg/mL) for 10 min in the dark and after incubation, at least 300 cells per sample were counted under a fluorescence microscope (Olympus IX71, Tokyo, Japan) using the total magnification of 200× (20× objective lens plus 10× eyepiece lens). The results were expressed as percentage of condensed nuclei—an average of three to five independent experiments, with one replicate per condition—and calculated according to the following equation:

For morphological analysis of myeloid cell differentiation, cytospins were prepared by centrifugation (1.200 rpm, 10 min) of 5 × 10⁴ primary AML cells on microscope slides (Paul Marienfeld GmbH, Lauda-Königshofen, Germany) using a Shandon Cytospin 3 Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, United States) and air drying for 5 min. Subsequently, cytospin slides were stained for 3 min with May-Grünwald (Merck, Darmstadt, Germany) and afterward for 13 min with 25% Giemsa solution (Merck). For each condition, 200 cells were analyzed for cellular morphology and counted by two individuals independently using a Leica DMLB light microscope type 020-519.511 (Leica, Wetzlar, Germany) and Color Video Camera model MC-3289 (Horn Imaging GmbH, Aalen, Germany).

Next, the adequacy of cytologic specimens (smears, cytospin) for nucleic acid–based testing was evaluated. Smear and cytospin specimens were compared with the reference samples. In case of smears, cell pellets were resuspended in approximately 10 µL PBS and pipetted onto a coated glass slide (microscope slides; Thermo Scientific). The samples were air-dried for up to 30 minutes before snap-freezing and storage at –80°C. Cytospin specimens were prepared by resuspending the cell pellets in approximately 300 µL PBS. The cytocentrifuge chamber was assembled by the sequential alignment of the cytofunnel (Shandon Single Cytofunnel with white filter cards; Thermo Scientific), a coated glass slide (Shandon Cytoslide; Thermo Scientific), and slide rack. The cell suspension was transferred into the cytofunnel and centrifuged for 5 minutes at 650g (Shandon Cytospin 3 cytocentrifuge; Thermo Scientific). Afterwards, the samples were air-dried for up to 30 minutes and snap-frozen.

At the stipulated time-points, hNECs were dissociated from Transwells with 1× Trypsin/EDTA solution (Gibco, Carlsbad, Calif). After fixation in 4% formaldehyde at room temperature for 10 min, and dual 1 × dPBS wash, single-cell suspension via cytospin was prepared at a density of 1 × 10⁴ cells/100 μl using Shandon Cytospin 3 Cytocentrifuge (Thermo Fisher Scientific) at 500 rpm for 5 min with mild acceleration.
The infected hNECs on the cytospin slides were permeabilized using 0.1% Triton X-100 at room temperature for 10 min, and washed three times with milli Q water. Cells were then blocked using 10% goat serum (DAKO, Glostrup, Denmark) for 30 min at room temperature, and incubated with primary antibody against MUC15 (1:50 dilution, Abcam) and HA (1:250, Abcam) in 1% goat serum (DAKO) at 4 °C overnight. After incubation, cells were washed three times with 1 × TBS, and incubated with fluorescent secondary antibody for 1 h at room temperature, followed by another three times of washing. Cells were then mounted using ProLong AntiFade reagent with DAPI and covered with microslides. Cytospin slides were observed using an Olympus IX51 fluorescence microscope under 40 × objective lens or 100 × oil lens.

After the removal of blood, the BALF was collected by instillation and suction of 0.9% normal saline (3 × 0.8 mL) in the lungs with 24-G intravenous catheter. BALF samples were centrifuged for 5 minutes at 400 xg at 4°C, and supernatants were collected and stored at -80℃ until use. The remaining cell pellets were used to prepare cytospin slides to identify cells present in the pulmonary air spaces. Using cytospin (Shandon Cytospin 3 Cyto-centrifuge, Thermo Fisher Scientific, Waltham, MA, USA), slides were prepared by centrifuging pellets at 1,165 xg for 10 minutes. After air-drying, cytospin slides were stained using H&E (Abcam, Cambridge, MA, USA) and Differential Quik stain (Diff-Quik, Polysciences, Inc., Warrington, PA, USA). Cytospin slides were visualized under a BX51 light microscope (Olympus, Tokyo, Japan).