Epilife media

HNSCC cell lines, UM-SCC-1 (SCC-1), UT-SCC-23 (SCC-23), UMSCC-5, and UMSCC-6, were sourced from the University of Michigan. UM-1 and UM-2 cell lines were procured from the University of California, Los Angeles, while normal human epidermal keratinocytes (NHEKs) were obtained from the ATCC. These HNSCC cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Normal human oral keratinocytes (NHOKs) and NHEKs were maintained in EpiLife media supplemented with human keratinocyte growth supplement (Invitrogen, Carlsbad, CA, USA). All cell lines were cultivated under standard conditions at 37°C in a humidified incubator with 5% CO₂. Hypoxic conditions were induced using an InvivO₂ 300 hypoxia workstation (Baker Ruskinn, Ltd., Bridgend, UK) flushed with nitrogen to establish an atmospheric composition of 94% N₂, 5% CO₂, and 1% O₂ at 37°C.

Normal human oral keratinocytes (NHOKs) were maintained in EpiLife media supplemented with the human keratinocyte growth supplement (Invitrogen, Carlsbad, CA, USA). UM1 and UM2 oral cancer cell lines
[22 (link)] were cultured in Dulbecco’s modified eagle medium (DMEM) plus 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). The cells were maintained at 37°C in a humidified 5% CO₂ incubator and passaged when they reached 90-95% confluence.

UM1 and UM2 oral cancer cell lines were cultured in the Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). Normal human oral keratinocytes (NHOKs) were cultured in EpiLife media supplemented with the human keratinocyte growth supplement (Invitrogen, Carlsbad, CA, USA). The cells were maintained at 37°C, 5% CO₂ in a humidified cell culture incubator and passaged when they reached 90–95% confluence.

Mouse BMDMs were isolated and differentiated as reported previously (20 (link)). In brief, WT or Trem2^−/− mice were sacrificed and the bone marrow in femurs was flushed out and cultured in DMEM containing 10% FBS and 30% (vol/vol) L-929 fibroblast-conditioned medium as a source of macrophage colony-stimulated factor. BMDMs were obtained as a homogeneous population of adherent cells after 7 days culture. The BMDM purity was routinely >95% as assessed by flow cytometry. A6(1) corneal epithelial cell line was a gift from Dr. Peggy Zelenka (National Eye Institute/NIH) and cultured following their protocol (24 (link)). The A6(1) cells were derived from corneal epithelia of the 14-day-old immortomouse (Charles River Laboratories, Wilmington, MA, USA), then conditionally immortalized by a temperature sensitive SV40 T-antigen, under the control of an IFN-γ inducible promoter. Cells were cultured in Epilife™ media, supplemented with corneal epithelial growth supplement, interferon IFN-γ (5 units/ml), 1% penicillin–streptomycin, 1% l-glutamine, and 20% (v/v) FBS (all from Invitrogen) at the permissive temperature of 33°C, in a humidified atmosphere of 95% air and 5% CO₂. For in vitro assays, subconfluent cultures were transferred to the nonpermissive temperature of 37°C and cultured in the same media, but without IFN-γ.

UMSCC-1 (SCC-1), UTSCC-23 (SCC-23), UMSCC-5, UMSCC-6, UM1, UM2, CAL-27, and FaDu cell lines were maintained in the Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U·mL⁻¹ penicillin, and 100 μg·mL⁻¹ streptomycin. Normal human oral keratinocytes (NHOKs) were cultured in EpiLife media supplemented with the human keratinocyte growth supplement (Invitrogen, Carlsbad, CA, USA). All cells were cultured at 37 °C in a humidified incubator with 5% CO₂.

Target cells were exposed for 24 h to different TPPs or solvent only as vehicle controls. Doses were chosen based on previous systematic dose- and time-dependent cytotoxicity studies [16 (link)]. For TPM and WS-CM, EC-30 doses were applied; for ST/CAS, the dose with the same amount of NIC as that in TPM at EC-30 was applied since no EC-30 could be determined. In addition, low and high doses of NIC alone were used as controls. The low dose (14 μg/mL) represented the level of nicotine in the combustible TPP exposures, while the high dose (474 μg/mL) was used to reach exposure level nearing the cytotoxic response in the TPP exposures. The high NIC dose was chosen in this range because it was previously shown that NIC at close to millimolar doses had some cytotoxic effects on other cell types [15 (link)] and since we had determined such range yielded 20% cytotoxicity in oral cells [16 (link)]. NIC vs. DMSO group comparisons were used mainly as controls for exposure and were not included for microarray expression profiling in the 101B cell line. Phenol Red-free media were used for these preparations (DMEM without Phenol Red for 101A, 101B cells; Invitrogen Epi-Life media for HGECs).

An overnight culture of Staphylococcus aureus ATCC 6538 in 5.0 ml of tryptic soy broth (TSB) was incubated statically at 37°C for 24 hours. Tissue culture inserts were placed in a 24 well plate and inoculated with 10 μl of overnight culture and 500 μl of TSB and inoculated at 37°C for 72 hours. Every 24 hours during that 72 hour period the TSB supernatant was removed, the inserts were moved to new wells in the 24 well plates, and 500 μl of fresh TSB was added to the wells. At the end of 72 hour period the TSB was removed and 500 μl of phosphate buffered saline pH 7.4 (PBS) was added and left for 1 hour to wash the remaining TSB from the tissue culture insert. After the removal of the PBS, 500 μl of Epilife media (Invitrogen, Carlsbad, CA) was added and incubated for 24 hours at 37°C. The new biofilm conditioned media was then removed from the well and filtered with a 0.45 μm syringe and collected in 15 ml centrifuge tubes. This BCM collecting and filtering procedure was repeated every 24 hours for 3 days. The collected BCM was then pooled and frozen at -20°C until it was needed.