Diff quik staining set

Viability of the negative control cells compared to the pre-miR Let-7b or si-LIN28B cells (hBMSC-Tum) was determined using an alamarBlue assay as previously described^{28 (link)}. All assays were carried out with the appropriate controls. Briefly, 1 × 10⁴ cells were cultured in 96-well plates and the cell viability measured at the indicated time points by adding 10% volume alamarBlue assay reagent and measuring the fluorescence at excitation and emission wavelengths of 530 nm and 590 nm, respectively.
The colony forming ability of the control cells compared to the pre-miR Let-7b or si-LIN28B cells (hBMSC-Tum), hBMSC-LIN28B cells, and hBMSC-mCherry cells was determined using a clonogenic assay as previously described^{29 (link)}. Briefly, cells were seeded into 12-well plates at different concentrations of serial diluted cells (1:2 to 1:64), with an initial seeding density of 1.5 × 10⁴ cells per well and incubated at 37 °C under 5% CO₂ for 10 d. The plates were then washed and stained using a Diff-Quik staining set (Siemens Healthcare Diagnostics). The plates were scanned and the number of colonies was evaluated under light microscopy.

Bone marrow-derived neutrophils were isolated from mice following the protocol of the Mouse Neutrophil Negative Selection Kit (STEMCELL Technologies Inc.). P. aeruginosa 8821 was opsonized with 10% mouse serum for 30 min at 37°C. The neutrophils were counted and then incubated with preopsonized P. aeruginosa 8821 (multiplicity of infection (MOI) = 10) at 37°C for 30 min. The neutrophil pellet was washed with PBS and then treated with PBS containing 0.1% trypsin and 0.02% EDTA for 15 min at room temperature. Neutrophils were resuspended in PBS containing 10% mouse serum. Specimens were prepared using the Cytospin^™ 4 Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA). The centrifuged specimens were then stained with a Diff-Quik^™ staining set (Siemens Healthcare Diagnostics Inc., Newark, DE) and examined under oil immersion. The number of bacteria engulfed by 100 randomly selected neutrophils was counted. The phagocytic activity was measured according to the rate of phagocytosis and the phagocytosis index. The rate of phagocytosis = number of cells containing bacteria/number of cells counted) X 100%. The phagocytosis index = total number of bacteria in all cells/ number of cells counted.