May gr nwald

ScreenCell^® filter morphology was assessed using May–Grünwald Giemsa staining. Filters were placed into a bath containing May–Grünwald (Sigma Aldrich Ltd., Dublin, Ireland) and incubated for 2 min 30 s with constant agitation, followed by a second incubation in a 1:1 dilution of May-Grünwald:dH₂O (pH 7) for a further 2 min with agitation. Filters were subsequently incubated in a stain consisting of a 1:10 dilution of Giemsa:dH₂O (pH 7) for 10 min. Excess stain was removed by washing in dH₂O (pH 7) for 1 min, and filters were allowed to air dry at room temperature for 20 min. Morphological assessment of stained filters was carried out using an Olympus BX41 light microscope (Olympus Life Science, Waltham, MA, USA). CTCs that were identified, based on morphology, were enumerated and recorded by an independent expert cytopathologist (B.H.). CTCs were defined as cells in the same plane of focus as filter pores whose nucleus was at least twice the diameter of a filter pore, dark blue/purple in colour and with an outline that was well-defined around its entire circumference. These criteria have been used for the morphological identification of CTCs in recently published studies of prostate cancer patients [27 (link)].

In the other set of mice, bronchoalveolar lavage was performed as previously described by our group [16 (link)]. Briefly, lungs were washed three times with 0.7 mL of sterile saline. Total cells were counted using an automated cell counter (LUNA-II, Logos Biosystems, France) and the BAL fluid was centrifuged at 1000× g for 10 min. The supernatant was stored at −80 °C until further analyses and the pellet was resuspended in PBS for differential cell counts. A volume of 100 µL of resuspended cells adjusted to 0.75 × 10⁶ cells/mL were spun at 450 rpm for 6 min (Cytospin™4 cytocentrifuge, Thermo Fisher Scientific, Waltham, MA, USA) onto microscope slides. Slides were air-dried, fixed in methanol for 10 min and stained with May–Grünwald (Química Clínica Aplicada S.A, Tarragona, Spain) for 5 min and 30% Giemsa (Merck, Darmstadt, Germany) for 15 min. Counts of macrophages, neutrophils, eosinophils, basophils and lymphocytes were carried out in a total of 400 cells from each sample in a blinded manner.