Haemocytometer

The RBC of fresh blood was assayed by Natt and Herrick's staining method, as detailed in Svobodová et al²⁵ . Briefly, 10 µl of fresh blood was mixed with 1990 µl of Natt and Herrick solution (Bioanalytic GmBH, Germany). Then 10 µl of this mixture was used for cell counts using a haemocytometer (Marien Feld, Germany) under a light microscope (Leica, Germany) with 40× magnification.

Viable cells were incubated at 37°C with 5% CO₂, and the medium was changed every 3‐4 days. Cells were observed on days 3, 7 and 14, using an inverted microscope (Olympus CK40, Melville, NY). Collected cells were resuspended in PBS and stained with trypan blue. The total number of viable cells was determined using a haemocytometer (Marienfeld, Germany).

All medium was removed and biofilms washed twice using diluted BHI. Biofilms were resuspended in 1 ml Hank’s balanced salt solution (HBSS) as described [13] (link). Briefly, bacterial suspensions (n = 3) underwent 10-fold dilutions in HBSS and 5×20 µl of each dilution spot plated onto CBA plates and incubated at 37°C/5% CO₂. For total cell counts (n = 3) bacterial suspensions were diluted 10-fold, stained with Syto 9 (Life Technologies, U.S.A.) according to manufacturer instructions, and individual cells counted using a haemocytometer (Marienfeld-Superior, Germany) and a Leica epifluorescent microscope using a 100x oil immersion lens.

For cell subculture, the media was discarded, and the cell culture flask was rinsed with 5 ml of DPBS -/-, detached by 2 ml of 0.05% of Trypsin-EDTA, and incubated at 37°C for 5 minutes before being neutralized with 8 ml of growth media. The solution was centrifuged at 1200 rcf for 6 minutes, and the cell pellet was resuspended with 2 ml of growth media. An aliquot of the growth media was used to determine the total living cell count by Trypan Blue dye exclusion method (Gibco Inc., Billings, Montana, USA) and counted using haemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, DE). After cell counting at each passage, the cells were seeded into three new T25 cell culture flasks with a cell-seeding density of 1500 cells/cm² and incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. The growth media was replaced every 3 days until the cell culture became subconfluent for subsequent subcultures. The cells were expanded until the working passage required for each experiment was achieved and the remaining cells were frozen for cryopreservation using freezing media containing 10 ml dimethyl sulfoxide (Sigma-Aldrich, St. Louis, Missouri, USA) and 90 ml of FBS in a cryovial tube which was then stored in liquid nitrogen.

A549 cells were harvested and counted using haemocytometer (Marienfeld, Germany). Then, cultured A549 cells (2.5 × 10⁶) subcutaneously (sc) implanted into the thigh of the right hind leg of 6-week-old mice. CB11 (5 or 10 mg/kg) was administered intraperitoneally (ip) for 4 days. To anesthetise, mice were injected intraperitoneally with Avertin solution before surgery. After surgery, mice were exposed with CO₂ in CO₂ chamber and maintain the CO₂ flow until the animal has stopped breathing.