Crystal violet assay

The cell invasion assay was performed according to previous methods described in detail elsewhere [17 (link),117 (link)]. Briefly, 5 × 10⁵ cells (treated with pan-PAD inhibitor Cl-amidine and the PAD isozyme-specific inhibitors with respective PBS or DMSO control as before) were plated on Matrigel-coated transwell filters (Corning™ BioCoat™ Matrigel™ Invasion Chamber with Corning™ Matrigel Matrix; BD Biosciences, Wokingham, UK) in a chemotactic gradient of 1:10% FBS. Following an incubation time of 16 h, the number of invaded cells was determined using the crystal violet assay (Abcam, U.K.) and the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Abcam, U.K.). The same number of cells was plated and incubated in parallel for 16 h, for assessment of PAD inhibitor-mediated effects on cell proliferation. The CLARIOstar plate reader (BMG Labtech, Aylesbury, UK) was used at 540–590 nm to measure absorbance and normalised according to the control. The experiments were performed in three biological and three technical repeats.

Five mice in each group underwent whole blood collection as above. One-hundred microliters of whole blood from each mouse were mixed with 100 μL of 1 × 10⁷S. aureus Xen36 CFU/mL for a final inoculum of 10⁶ CFU in 200 μL. This solution was added to each well within a 96-well-flat bottom plate. Six additional control wells containing 200 μL of saline were also included for standardization. After 24 h of incubation at 37°C, each well was washed with PBS three times to remove residual blood cells and non-adherent bacteria. A well-validated crystal violet assay (Abcam, Cambridge, United Kingdom) (62 (link)–64 (link)) was performed to quantify the biomass of the residual biofilm formation by OD at 595 nm.

Six mice in each group underwent whole blood collection as above. 100 μL of 1 x 10⁷S. aureus Xen36 CFU/mL and 100 μL of blood from each mouse were mixed into each well of a 96-well flat bottom plate. Additionally, 200 μL of saline was used as a standardized control. The plate was incubated for 24 hours at 37°C (MaxQ 4450; ThermoFisher Scientific) to permit sufficient biofilm formation. Wells were then washed with PBS three times to remove blood and non-adherent bacteria. In order to quantify mass of the residual biofilm, a well-validated crystal violet assay (Abcam, Cambridge, United Kingdom) was performed and absorbance was measured by OD at 595nm (FLUOstar Omega, BMG Labtech, Ortenberg Germany). Values were reported as absorbance units.