Diff quick

5 × 10⁵ cells/ml were seeded in Matrigel invasion chamber (8.0 μm pore size; BD Biosciences, 354480). Top chambers (culture inserts) were filled with serum‐free medium, and bottom chambers were filled with medium containing 10% FBS. After 24 h, cells at the upper membrane surface were removed with a cotton swab. Cells on the lower side of the filters were fixed, stained with Diff quick (Sigma‐Aldrich), photographed, and at least 6 fields were counted. Each experiment was performed for three times, in triplicates. For CAF‐induced cancer cell invasion, cancer cells were pre‐treated or not with CM derived from control and/or hMENA(t) silenced CAF for 48 h, and then, invasion assays were performed as described above. For Matrigel invasion assay toward GAS6, recombinant GAS6 (200 ng/ml) were added into the lower chamber. Each group was plated in duplicates in each experiment, and the experiment was repeated at least 2 times.

Chemotaxis of bone-marrow-derived cells was performed as described previously (27 (link)). Briefly, bone-marrow neutrophils were collected as described above and then resuspended in chemotaxis medium (HBSS containing 2% BSA) at a density of 5 × 10⁶ cells/ml. As a chemoattractant, we used C5a (12.5 nM; Hycult, Uden, Netherlands), which was diluted in chemotaxis medium. The chemoattractant was placed in the bottom wells of a modified Boyden chamber (Neuro Probe, Gaithersburg, MD, USA) and overlaid with a 3 µm polycarbonate membrane. Then, 50 µl of the cell suspension were placed in the top wells of the assembled Boyden chamber and incubated at 37°C in 5% CO₂ for 30 min. Subsequently, the membranes were removed and the cells on the bottom of the membrane stained with Diff-Quick (Merck, Darmstadt, Germany). The numbers of migrated cells for each well were counted in five different high-power fields and the number of cells per mm² was calculated by computer-assisted light microscopy. Results are expressed as the mean value of triplicate samples of at least two independent set of experiments.

Bronchoalveolar lavage (BAL) was performed 24 h after the last OVA challenge as previously described [21 (link)]. Mice were anaesthetized i.p. (ketamine 150 mg/kg, xylazine 10 mg/kg). A plastic cannula was inserted in the exposed trachea, and airways were washed with 0.5 mL of 0.9 % NaCl (saline) injected with a 1 mL syringe. This procedure was performed ten times. The initial concentrated supernatant of the two first lavages (2 × 0.5 mL administered, approx. 0.5 mL recovered) was collected for cytokine measurements. The remainder of the BALF was centrifuged (600 g for 5 min, 4 °C), and cell pellets pooled. After lysis of erythrocytes with distilled water followed by osmotic re-equilibration and centrifugation, the cell pellet was suspended in 500 μL of saline and total cell counted on a haemocytometer chamber (Neubauer, PRECISS®). Cells were cytocentrifuged at 700 rpm for 10 min (Cytospin 4, Thermo Fischer Scientific), and stained with Diff-Quick® (Merck) for differential cell counts performed on at least 400 cells.

Peripheral blood samples were obtained from anesthetized mice by inferior vena cava puncture using a 26 gauge needle and 1 mL syringe containing heparin sodium (Sigma Aldrich, St. Louis, USA). Complete blood counts (CBC) and differential white blood cell (WBC) counts were done using an ADVIA 2120i Hematology System (Siemens Healthcare, Erlangen, Germany). Blood smears were stained with diffQuick (Merck, Darmstadt, Germany).

All vaginal samples were collected from the middle vaginal vault after disinfection of the perivulvar area with povidone-iodine and through a sterile speculum. Two swabs were collected for metataxonomic analysis (Fecal Swab, Copan Italia, Brescia). A third swab was collected for bacteria culture (Transystem, Copan Italia, Brescia), and a fourth swab was collected for cytological examination.
The samples for metataxonomic analysis and culture were maintained at 4 °C for a maximum of 36 h before being processed [30 (link), 31 (link)]. The swab for cytology was gently rolled over a microscope slide, air dried and stained with Diff Quick (Merck S.p.A., Milano, Italy).