Defined keratinocyte serum free media

Immortalized TERT-2/OKF-6 (BWH Cell Culture and Microscopy Core, Boston, MA, USA) oral keratinocytes from non-cancerous tissue from the floor of a human mouth were cultured in defined keratinocyte serum-free media (Gibco, Waltham, MA, USA) with 1% penicillin/streptomycin (Gibco) under standard cell culture conditions [44 (link)].

Eyelids were explanted from mid-to-late E15.5 embryos into warm media (Defined Keratinocyte Serum-free Media supplemented with 600 μm calcium and 5% w/v penicillin-streptomycin, Life Technologies). For live imaging, we used methodology previously described for embryonic skin explants (Li et al., 2011 (link)). Using a small volume of growth factor-reduced matrigel, allowed to polymerize for 25 min at 37°C, we sealed eyes against a Lumox teflon-bottom dish (Sarstedt). Eyelid closure was imaged for periods of 6–16 h in 5% CO2 on a PerkinElmer Volocity spinning disk system equipped with a heated enclosure and gas mixer (Solent) and ×20/0.75 CFI Plan-Apo objective, or a Zeiss LSM 780 system with a stage-top incubator and Plan-Apochromat ×20/0.8 objective.

Healthy gingival tissue was obtained from healthy human subjects by oral surgery to produce primary GEC cultures as previously described (Yilmaz et al., 2004 (link)). With the consent of patients, gingival epithelial tissues were collected under the approval of the Institution Review Board of the University of Florida. GECs were cultured as monolayers in serum-free keratinocyte growth medium (Lonza) at 37°C in a 5% CO2 incubator. The human immortalized GEC cell line was obtained as previously described (Oda et al., 1996 (link)) and grown in defined keratinocyte serum-free media (Life Technologies) at 37°C in a 5% CO2 incubator. Prior to F. nucleatum infection, media was changed to Opti-MEM media (Life Technologies).