Six well plate

SCAPs were plated onto six-well plates (Costar, USA) and cultured at 37°C in a CO₂ incubator. When cells reached 70% confluence, centrifugal force was applied by a centrifuge (Allegra 64R, Beckman Coulter, USA) equipped with specially designed rotors to hold six-well or ninety six-well culture plates. The strain experienced by the cells in the culture plates was provided through a top-bottom axis (Figure 1(a)). Actually the force (F) is decided by the centrifugal radius of the centrifuge (R, in cm) and rotates speed (RS, revolutions per min). Consider F = 1.18 × 10⁻⁵ × R × RS² (Figure 1(a)). Then SCAPs were cultured in the plates and centrifuged at 200 g (corresponded to a speed at 1000 rpm) for 30 min [15 (link)], which resembles the clinical orthodontic force, and then returned to the incubator. Control cells were cultured in absence of centrifugal loading. Proliferation and differentiation of SCAPs were assessed at different time points.

Cells were grown in six‐well plates (Costar, Cambridge, MA, USA) and transfected with a mixture of 30 nmol/ml MXRA5 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Opti‐MEM I Reduced Serum Medium and Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Life technologies, Paisley, UK). After 18 hr, cells were washed and cultured for 48 h in complete medium and serum‐depleted for 24 hr before stimulation. A scrambled siRNA (Ambion, Applied Biosystems, Foster City, CA, USA) was used as control.

Cells were grown in six-well plates (Costar) and transfected with a mixture of 20 nmol/ml BASP1 siRNA (Ambion, Applied Biosystems), Opti-MEM I Reduced Serum Medium and siPORT Amine Transfection Agent (Ambion, Applied Biosystems).^{19 (link)} After 18 h, cells were washed and cultured for 48 h in complete medium and serum-depleted for 24–48 h before stimulation. These time points were selected from a time-course of BASP1 protein expression in response to siRNA. A negative control scrambled siRNA provided by the manufacturer did not reduce BASP1 protein.

A murine macrophage cell line RAW264.7 obtained from American Type Culture Collection (ATCC, Rockville, MD) was cultured in DMEM supplemented with 4500 g of glucose/L, 110 mg of sodium pyruvate/L, 1% penicillin–streptomycin (Sigma-Aldrich, UK), and 10% heat-inactivated fetal bovine serum (FBS) (J R Scientific, Inc., USA). The cells were cultured at 37°C in a 5% CO₂ atmosphere incubator (NuAire, Plymouth, MN, USA). The cells were subcultured when at 80% confluence, and the cell suspension was diluted to 5 × 10⁵ cells/mL for the experiments [19 ]. One hour before the experiments, 1 mL of fresh medium (37°C) was placed on the six-well plates (Costar, Corning, NY). The cells were cultured for 24 h.

For this assay, dual-species biofilms were grown in the six-well plates (Costar) containing 4 mL of the microbial suspension, as described above. After 96 h with the solutions, the biofilms were resuspended in 0.85% NaCl, scraped from the wells, and the liquid phase of the extracellular matrix was extracted by sonication (for 30 s at 30 W), as detailed elsewhere [47 (link)]. The bicinchoninic acid method (Kit BCA; Sigma-Aldrich) was performed for protein determination of the extracellular matrix, using bovine serum albumin as the standard [47 (link)], while the carbohydrate content was measured using the method devised Dubois et al. [48 (link)], with glucose as the standard. For DNA content, a volume of 1.5 mL of the liquid phase of the extracellular matrix was spectrophotometrically analyzed (at 260 and 280 nm) in a Nanodrop Spectrophotometer (EONC Spectrophotometer of EONC, Biotek, Winooski, VT, USA) [27 (link)]. Protein, carbohydrate, and DNA values were expressed as mg g dry weight of biofilm.

For growth curve assays, 7 × 10³ cells/cm² were plated (except when differently stated) in Costar^® six-well plates. Forty-eight hours later, a six-well plate was collected to determine P0. This procedure was done to eliminate the lag phase of these cells and observe the effects of treatment and/or induction in the exponential phase. For maximum cell density saturation assays, 20 × 10³ cells/cm² were plated. After 10 days (P10), the cultures reached their saturation density. The induction using 4-hydroxytamoxifen (4OHT) was initiated in P0, for the growth curve, and P10, for cell saturation density assays. In the days indicated cells were harvested using trypsin and resuspended in PBS + 5% fetal bovine serum (FBS) to inactivate the trypsin, washed, and fixed in a solution of phosphate buffer (PBS) with 3.7% of formaldehyde. All treatments and fresh medium were changed every other day. The cells were counted in Beckman Counter Z2^®. The graphs present the results of at least two biological replicates in which three technical replicates for each treatment were collected.