Crystal violet

Following the migration assay of SaOS-2, SaOS-LM2, and HT-1080, transwell supports were washed twice with Dulbeco’s phosphate-buffered saline (Gibco, Cat.# 14190144) and cells that did not migrate were removed from the upper chamber using a moistened cotton swab. Migrated cells that adhered to the lower surface of the support were stained with crystal violet (Fisher Scientific, Cat.# C58125) for 10 min. Transwell supports were washed with Dulbeco’s phosphate-buffered saline three times and air-dried. Once dry, bright field images were taken using Olympus cellSens Dimension software (Version 2.1) on an Olympus SZX16-ILLT microscope (Olympus, Tokyo, Japan) with 5× objective lens and 2.9× zoom. The crystal violet bound to migrated cells was eluted from the supports by pipetting 400 µl of 33% acetic acid (Fisher Scientific, Cat.# A38500) into each upper chamber and shaking the plates for 10 min. Half of the eluent for each sample, 200 µl, was transferred to a 96-well clear microplate (Corning, Cat.# 3595) and the absorbance at 590 nm was determined using the Tecan Infinite M200 plate reader (Tecan Group Ltd., Männedorf, Switzerland) and Tecan i-control software (Version 3.91.0). Standard curves were generated for each cell line used in the migration assay and were used to calculate the cell concentration from the experimental absorbance measurements.

The effect of different Fabs on S. mutans and S. sobrinus biofilm formation were examined using the microdilution method^{41 (link)}. Briefly, overnight culture of S. mutans and S. sobrinus were prepared to a final concentration of 10⁶ CFU/mL and grown in BHI for 24 hrs at 37 °C in a 5% CO₂ aerobic atmosphere with the addition of 1% sucrose for biofilm development. Different Fabs were added to the culture at 0 hour at a final concentration of 3 μM. After 24 hours, before biofilm quantification, the culture growth was measured at OD_600nm using a plate reader. The cell suspension was carefully removed and each well was washed three times with 1× PBS. The remaining attached cells were fixed with 96% ethanol for 15 min and then plates were dried. Wells were stained with 120 μL of 0.1% crystal violet (Fisher) for 20 minutes. The crystal violet stain was aspirated and wells were washed three times with 150 μL of PBS. Wells were air-dried and retained crystal violet was solubilized with 150 μL of 30% (v/v) glacial acetic acid (Fisher). The plate was incubated at room temperature for 20 min with shaking. The biofilm formation was quantified by measuring the OD_590nm using a plate reader.