Maxq 4000 orbital shaker

For the 6 well plate method, a 22 × 32 mm coupon was inserted at an angle into each well of a sterile polystyrene 6-well microplate (ThermoFisher) to which was added 10 mL of growth medium and a 100 µL of overnight culture (Fig. 1). For the Duran bottle method, a polyurethane bung was inserted into a 100 mL Duran Bottle and sterilised. Medium and a 1 in 100 dilutions of the overnight culture was added to the Duran Bottle to a final volume of 70 mL, and a sterile slide was inserted into the bottle. Plates or Duran bottles were incubated in a MaxQ 4000 orbital shaker (Thermo Scientific, Paisley, UK) with an orbit of 19 mm at 30 °C and 70 rpm for 3 days.Fig. 1

Schematic of a 6-well plate and b Duran bottle methods of biofilm formation

CS/DS disaccharides were isolated and quantified according to our previously published methods (Alonge et al., 2019 (link), 2020 (link); Logsdon et al., 2021 (link)). Five PFA-fixed coronal brain sections (30 μm) from each mouse were chosen for CS/DS disaccharide extraction and quantification. Sections were washed 3× in Optima LC/MS-grade water and 1× in 50 mM ammonium bicarbonate (pH 7.6) at RT. Chondroitinase ABC enzyme (ChABC, Sigma, C3667), a combinatorial endo- and exo-lyase that selectively degrades all CS/DS-GAG chains into their individual disaccharide units (Yamagata et al., 1968 (link); Hamai et al., 1997 (link); Prabhakar et al., 2009 (link); Spliid et al., 2021 (link)), was reconstituted (500 mU/ml) in 50 mM ammonium bicarbonate (pH 7.6) to digest CS/DS-GAGs at 37°C in a Thermo Scientific MaxQ4000 orbital shaker for 24 h. Supernatants were collected in sterile 1.7 ml microcentrifuge tubes and spun for 10 m at 14k× g to pellet any debris. The supernatant was then dehydrated using a SpeedVac Concentrator, and the lyophilized product was reconstituted in 30 μl of LC/MS-grade water.

A single E. faecalis OG1RF colony was inoculated into 5 mL of BHI containing 50 μg ml⁻¹ rifampicin and incubated overnight with shaking at 37°C in a MaxQ 4,000 orbital shaker (ThermoFisher, Waltham, MA). The stationary phase culture was diluted (1:10 [vol./vol.]) into fresh media and, and the absorbance at 600 nm was monitored using a Biomate™ 3 Series spectrophotometer (ThermoFisher, Waltham, MA) until mid-log phase growth. Fifty microliters of the mid-log phase E. faecalis OG1RF culture was plated using autoclaved borosilicate glass beads (ThermoFisher, Waltham, MA) on brain heart infusion agar plates containing 50 μg ml⁻¹ rifampicin and 100 μg ml⁻¹ streptomycin and incubated overnight at 37°C in a Steri-Cult CO₂ incubator (ThermoFisher, Waltham, MA) for 24 h.

After the TBI, the supernatant of tube 1 was collected in tube 2 using a magnetic separator (Qiagen, Germantown, MD, USA). The concentration of the supernatant was determined using a NanoDrop 2000 UV-Vis Spectrophotometer and confirmed that the concentration decreased consistently by a factor of 11 (2.2 mg/mL to 0.2 mg/mL Tyr) due to Tyr binding to MB. Subsequently, 1 mL of wash buffer (10 mM Tris-HCl, pH of 8.0, 5 mM imidazole, 250 mM NaCl, 0.05% Tween-20) was pipetted into tube 1 twice and the wash was collected into tubes 3 and 4 each time. The MB were washed with 1× PBS a third time afterward. The MB were then resuspended with 100 µL of 1× PBS in tube 1. This was followed by adding 100 µL of the 3 mM L-DOPA solution (1:1 ratio), as described in the colorimetric activity measurement method below, into tubes 1, 2, 5, and 6. These tubes were immediately followed with the post-binding Tyr-MB incubation in a MaxQ 4000 Orbital Shaker (Thermo Scientific, Waltham, MA, USA) for 30 min (270 rpm) at each temperature condition of 5, 25, 31, 37, and 43 °C, or MB volume condition at 50, 100, 150, and 200 µL.