BALF was obtained by use of a technique adapted from Caldow et al.[47] . A sterilized 100 cm BAL catheter was inserted through a naris and blindly guided through the nasal passage into the trachea until the end was wedged in a bronchus. Once wedged in the appropriate location, a syringe was connected to the catheter and a total of 30 mL sterile saline (37 °C) solution was slowly infused and immediately aspirated back into the syringe after each infusion. BALF (17.7 ± 0.4 mL) was obtained from each calf and stored in a 50 mL tube on ice until further processing in the lab the same day.
Thereafter, BALF was filtered by passing through a 70 μm cell strainer (Corning, NY) to remove debris. To obtain cell pellets and perform cell counts, BALF suspension was centrifuged (5 min, 400 × g at 4 °C) and the remaining pellet was re-suspended in 1 mL cold fetal bovine serum (FBS; 4 °C). After centrifugation, the supernatant was aliquoted into 1.5 mL tubes and stored at −80 °C for further analysis. Cell number was determined by automatically counting in a Cellometer Bright Field cell counter (Nexcelom Bioscience, Lawrence, MA). For differential BALF cell counts, 0.5 × 106 BALF cells were used to make cytospins stained with Diff-Quick (Medion Diagnostics, Medion Diagnostics International Inc., Miami, FL) and a minimum of 400 cells were counted.
Thereafter, BALF was filtered by passing through a 70 μm cell strainer (Corning, NY) to remove debris. To obtain cell pellets and perform cell counts, BALF suspension was centrifuged (5 min, 400 × g at 4 °C) and the remaining pellet was re-suspended in 1 mL cold fetal bovine serum (FBS; 4 °C). After centrifugation, the supernatant was aliquoted into 1.5 mL tubes and stored at −80 °C for further analysis. Cell number was determined by automatically counting in a Cellometer Bright Field cell counter (Nexcelom Bioscience, Lawrence, MA). For differential BALF cell counts, 0.5 × 106 BALF cells were used to make cytospins stained with Diff-Quick (Medion Diagnostics, Medion Diagnostics International Inc., Miami, FL) and a minimum of 400 cells were counted.