48 well tissue culture plate

HeLa 57A and HEK293 cells were grown in 48-well tissue culture plates (Corning, Schiphol-Rijk, The Netherlands) in culture medium up to 50–60% confluency. Transient transfection with the plasmids carrying the indicated genes was performed with FuGENE 6 (Roche Diagnostics, Almere, The Netherlands) or FuGENE HD (Promega, Leiden, The Netherlands) according to the manufacturer’s instructions. A 3:1 ratio of FuGENE:DNA was used. The total amount of plasmid DNA per transfection was 250 ng/well. For transfection of HT-29 cells, adherent HT-29 cells at a confluence of ~80–90% were trypsinized and re-plated in 48-well tissue culture plates. Directly after plating, cells were transiently transfected with ExGen 500 (Fermentas GmbH, Germany) according to the manufacturer’s instructions. A 6:1 ratio of ExGen 500:DNA was used. The total amount of DNA used per transfection was 500 ng/well. Forty-eight hours post-transfection, the medium was refreshed with new culture medium (including FCS). Cells were pre-treated for 30 min with 10 mM Na-Bu, 10 mM Na-Pro or 2 μM TSA or left untreated. After the pre-incubation period, the medium was not refreshed prior to stimulation with the TLR agonists or TNFα unless indicated otherwise. Pre-treated or untreated cells were stimulated for 5 h with the TLR ligands or TNFα.

The human monocytic cell line, THP-1 (ATCC, Manassas, VA), was maintained in complete medium comprised of RPMI 1640 (Sigma-Aldrich) supplemented with 10% FCS (GE Healthcare Life Sciences, South Logan, UT) and 1% Penicillin-Streptomycin (Wako Pure Chemical) in a 5% CO₂ humidified atmosphere at 37 °C.
For differentiation to a macrophage phenotype, THP-1 cells (1.1 × 10⁵ /well) were incubated with 100 nM PMA in 48-well tissue culture plates (Corning, Kennebunk, ME) at 37 °C for 2 days. Following differentiation, PMA-containing media was replaced with complete medium and cells were rested for 24 h.

β-TCP scaffolds were transferred to 48-well tissue culture plates (Corning, Wiesbaden, Germany), and hMSCs embedded in the collagen I/III gels or directly seeded on the β-TCP scaffolds were kept in culture for 8 weeks. mBMSCs embedded in gels were pre-expanded for 1 week, and c-kit⁺ cells were seeded and co-cultures kept at 37 °C in a 20 %-O₂/5 %-CO₂-humidified atmosphere. Cells were recovered by vigorous pipetting and subsequent gel digestion using collagenase II (Life Technologies) for collagen I/III gels and dispase II (Life Technologies) for Matrigel® gels.

Biofilms that have been formed in 48-well tissue culture plates (Corning) following the incubation of 500 μL S. mutans in BHI with 2% sucrose in the absence or presence of CBG or respective ethanol concentrations were washed twice with 500 μL PBS followed by lysis in 200 μL of 0.04M NaOH for 1 h at 60 °C in a water bath under constant shaking. At the end of incubation, 18.5 µL of 1M Tris-HCl pH 7 were added to neutralize the NaOH [37 (link)]. The amount of DNA in each sample was quantified by qPCR with specific primers for S. mutans 16S rRNA (Table 1) using Power Green Master Mix (Applied Biosystems, Waltham, MA, USA). The PCR amplification was performed in a Bio-Rad CFX Connect Real-time system with the Bio-Rad CFX Maestro program. The amount of DNA was quantified according to the standard curve obtained using known DNA concentrations of purified S. mutans DNA. Purified DNA was extracted from an overnight culture of S. mutans using GenElute Bacterial Genomic DNA kit (Sigma Aldrich, St. Louis, MO, USA) as per the manufacturer’s instructions.

Human epidermal cells (HaCaT) (provided by H. Diggelmann, ISREC, Switzerland) were grown to confluence on 48-well tissue culture plates (Costar) [61 (link)]. Each well was inoculated with approximately 30 PFU of HSV-2 strain MS. Virus infection was allowed to proceed for 1 h at 37°C, then virus that had not entered cells was removed by citric acid wash at pH3.0 for 1 min at RT followed by a PBS wash. Serial 4-fold dilutions ranging from 40 μg/ml to 0.67 μg/ml of IgG purified from MAbs DL6, MC14, MC16, and from rabbit anti-gE2 polyclonal serum R265 were added to wells in 250 μl of DMEM containing 5% fetal bovine serum and incubated at 37°C in 5% CO₂ for 48 h [32 (link)]. Monolayers were stained with crystal violet and plaque size measured using an inverted microscope fitted with an eyepiece micrometer [61 (link)].