May gr nwald and giemsa solutions

Monocytes were isolated from cryopreserved PBMCs as described above and cultured for 5 d in complete RPMI with 10% FBS at 10⁶ cells/ml in the presence of 100 ng/ml M-CSF, 40 ng/ml of IL-4 (Miltenyi Biotec), and 5 ng/ml TNF (R&D Systems). After culture, cells exhibiting the macrophage or DC phenotype were quantified using flow cytometry staining for CD16 (clone 3G8; BioLegend) or CD1a (clone HI149; BioLegend) markers, respectively. CD163 (clone GHI/61; BioLegend) and viability dye (DAPI) were also included in the staining panel, and the staining buffer was composed of PBS with 0.5% human serum and 2 mM EDTA. For morphological analysis, cells were cytospun, stained with May–Grünwald and Giemsa solutions (Sigma-Aldrich), and imaged using a CFW-1308C color digital camera (Scion Corporation) on a Leica DM 4000 B microscope (Leica Microsystems).

A549 ATCC^® CCL-185™and NCI-H460 [H460] ATCC ^® HTB-177™ lung cancer cell lines were kindly provided by the European Institute of Oncology in Milan. Cells were propagated according to the instructions provided by the American Type Culture Collection (Manassas, VA, USA). Before performing spike-in experiments, the cells were detached with Trypsin-Versene^® solution (Lonza, Basel, Switzerland), counted using the Trypan Blue 0.4% solution as a vital dye exclusion assay (viability >99% in all tests) and injected in 10 mL of blood collected from healthy volunteers at a dilution of 1000 cells per milliliter of blood (i.e., 1000 cells per membrane spot). The spiked-in samples were incubated for 30 min at room temperature under gentle agitation until chemical fixation and filtration as described before. The membranes were stained with Hematoxylin (Histo-line Laboratories Srl, Milan, Italy) for 3 min and Eosin Y aqueous solution (Histo-line Laboratories Srl) for 1 min, or with May–Grünwald and Giemsa solutions (Sigma) as described before and observed at the Olympus BX51 under a 20× magnification objective.

A droplet of each blood sample was used to prepare blood smears, stained by the Pappenheim method with May-Grünwald and Giemsa solutions (Sigma-Aldrich). Blood morphology was recorded under a light microscope (DM750; Leica Microsystems GmbH, Wetzlar, Germany) connected to a digital camera, using LAS EZ v 2.0 software (Leica). Total white blood cells were counted and shown as the % of all counted cells in blood samples.