Leica application suite acquisition software

BALF collection was performed with the method of Shin et al., (2015) (link). Briefly, to obtain BALF, ice-cold PBS (0.7 ml) was infused into the lungs two times and withdrawn each time with a tracheal cannula (total volume, 1.4 ml). The collected BALF was centrifuged at 1,000 rpm for 10 min at 4°C. The supernatants were collected and stored at −70°C before cytokine analysis. The cell pellet was re-suspended with 500 μL ice-cold PBS and attached on a slide using a Cytospin 4 centrifuge (Thermo Scientific, Waltham, MA, USA) (1,000 rpm, 5 min, 20°C). Differential cell count was performed with Diff-Quik® staining reagent according to the manufacturer’s instructions. Five images of each slide were captured with a Leica DM5000B microscope and the Leica Application Suite acquisition software (Leica Microsystems, Wetzlar, Germany) under 40× objective lens. Thereafter, total cells, eosinophil, macrophages, and other cells (neutrophils and lymphocytes) were counted. The levels of IL-4, IL-5, IL-6, IL-13, eotaxin (R&D system), and MUC5AC (Cusabio Biotech Co.) in BALF supernatant were measured using ELISA kits, according to the manufacturer's instructions. The absorbance was measured at 450 nm using a microplate reader (iMark^TM, Bio-Rad Laboratories, Richmond, CA, United States).

Differentiated PLB-985 WT, PLB-985 X-CGD, and PLB-985 NCF1 ΔGT cells, as well as transduced PLB-985 NCF1 ΔGT cells were incubated in growth medium supplemented with 100 μg/mL NBT in the presence of 200 ng/mL PMA at 37 °C and 5% CO₂ for 30 minutes. Subsequently, the cells were fixed in 1% (vol/vol) formaldehyde. 250 cells per cytospin slide were analyzed manually for NBT activity using a Leica DM IL Fluo light microscope equipped with a DFC420 digital camera and LEICA application suite acquisition software (Leica Microsystems AG, Glattbrugg, Switzerland).