Casy automated cell counter

K562 cells were seeded at a density of 50,000 cells/ml in a 12-well plate. Indicated treatments were added to separate wells (three wells per condition). Cell counts were obtained at 24 h intervals using the CASY automated cell counter (Roche). The proliferation rate was determined based on the following formula: $\mathrm{Proliferation}\mathrm{rate}\left(\mathrm{doublings}\mathrm{per}\mathrm{day}\right)={\log }_2\left(\mathrm{final}\mathrm{cell}\mathrm{count}/\mathrm{initial}\mathrm{cell}\mathrm{count}\right)/\mathrm{no}.\mathrm{of}\mathrm{days}.$

The isolation of PEC, siLP and mLN was performed as previously described (4 (link)). For the isolation of lung cells, the chest cavity was opened via incision and the whole lung was perfused with 20mL ice-cold PBS via puncture to the heart using a 27G needle and syringe. Each lung was then removed and manually minced into small pieces. The minced tissue was then transferred to a 50mL falcon tube containing 10mL digestion medium (150µg/mL collagenase D and DNase I in PBS). The samples were incubated for 1 hour at 37°C under continuous shaking at 250rpm. Following the digestion step, the lung tissue was passed through a 70µm cell strainer into a fresh 50mL falcon tube. After erythrocyte lysis, the lung samples were washed and resuspended in cRPMI. All cell suspensions were counted using a CASY automated cell counter (Roche-Innovatis, Reutlingen, Germany).

HL-60 cells (ATCC; CCL-240) were grown in an incubator at 37°C (95% humidity, 5% CO₂) in Iscove's Modified Dulbecco's Medium (IMDM) (PAN-Biotech) containing 20% FBS (Biochrom) in polystyrene tissue culture flasks (Sarstedt). Cells were seeded at a density of 0.3×10⁶ cells per ml and 50% of the medium was replaced once per week. Confluent cultures were split 1∶3 to 1∶4 once per week.
Differentiation experiments were performed in Cell⁺ tissue culture flasks (Sarstedt). 1 μM working solutions of Vitamin D₃ (VitD₃)and all-trans retinoic acid (ATRA) were prepared in ethanol. Cells were seeded at 0.3×10⁶ cells per ml. Monocytic differentiation was achieved in macrophage serum-free medium (SFM) supplemented with M-CSF (50 ng/ml) and 0.5 μM VitD₃. Granulocytic differentiation was carried out in macrophage SFM with GM-CSF (10 ng/ml) and 0.5 μM ATRA. Cells grown in the respective medium containing 0,05% ethanol were used as controls. Cells were harvested by scraping with a rubber policeman and centrifugation for 5 min at 500 g at room temperature. They were finally washed once with PBS. Cell number was determined by counting in a Casy automated cell counter (Roche). Differentiation phenotype and viability were monitored by flow cytometry and Pappenheim staining. Experiments were repeated in two biological replicates.