Six human oral squamous cell carcinoma (OSCC) cell lines and normal human oral fibroblast (NHOF) cells used in this study were obtained from Professor Ian Charles Paterson, Department of Oral Biology and Biomedical Sciences, Faculty of Dentistry, University of Malaya. The derivation and culture of the OSCC cell lines have been described previously [13 (link)]. Briefly, OSCC cell lines were cultured in T-75 cm2 culture flask (Corning, USA) containing DMEM/F-12 (Nacalai Tesque, Japan), supplemented with 10% (v/v) fetal bovine serum (FBS) (JR Scientific, USA). Normal human oral fibroblasts (NHOF) were cultured in DMEM (Gibco, USA) supplemented with 20% (v/v) FBS. Cells were grown at 37°C with 5% CO2 in a humidified atmosphere.
Dmem f 12
Manufactured by Nacalai Tesque
Sourced in Japan, United States
DMEM/F-12 is a cell culture medium commonly used in research applications. It is a balanced salt solution that provides essential nutrients, vitamins, and amino acids required for the growth and maintenance of various cell lines in vitro.
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Lab products found in correlation
Axiovert 200m microscope, by Zeiss (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Nacalai Tesque (4 mentions)
Penicillin, by Nacalai Tesque (4 mentions)
Streptomycin, by Nacalai Tesque (4 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Htert rpe1 cells, by American Type Culture Collection (3 mentions)
Horse serum, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Fujifilm (3 mentions)
Streptomycin, by Fujifilm (3 mentions)
Non essential amino acids (neaa), by Nacalai Tesque (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Jmp pro 13, by SAS Institute (2 mentions)
Insulin, by Merck Group (2 mentions)
X tremegene 9 dna transfection reagent, by Roche (2 mentions)
Jmp pro 12, by SAS Institute (2 mentions)
Rpmi 1640 medium, by Nacalai Tesque (2 mentions)
Hela cell, by American Type Culture Collection (2 mentions)
38 protocols using dmem f 12
1
Culturing Human Oral Cell Lines
2
hPSC Culture and Maintenance Protocols
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Two different hPSCs were used in this study: H9 (WA09; passage 35) was purchased from the WiCell Research Institute (Madison, WI, USA) and CHA-SCNT-PSC-18 (Korea Stem Cell Registry code hES12019001), which we previously established [33 (link),34 (link)]. H9 cells were maintained in the mTeSR medium (Stem Cell Technologies, BC, Canada). CHA-SCNT-PSC-18 was cultured on mitotically inactivated mouse embryonic fibroblasts (CF1; Jackson Laboratory, CA, USA) in Dulbecco’s modified Eagle medium (DMEM)/F12 (Nacalai Tesque, Kyoto, Japan) supplemented with 20% knockout serum replacement (KSR; Thermo Fisher Scientific), 0.1 mM beta-mercaptoethanol (Thermo Fisher Scientific, MA, US), 1% non-essential amino acids (NEAA; Thermo Fisher Scientific), and 4 ng/mL recombinant human FGF2 (Gibco). The hPSCs medium was changed daily, and cells were passaged every 4–5 days. The hPSCs were maintained on Matrigel (BD Biosciences, San Jose, CA, USA)-coated plates in mTeSR and passaged when reaching 70–80% confluency. Cells before passage 45 were used to generate hSkMOs.
3
Differentiating Human iPSCs into EBs
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SNL feeder cells cultured with human iPSCs were eliminated by treatment with CTK
solution: 0.25% (v/v) trypsin, 0.1 mg/mL collagenase IV, 1 mM CaCl2, and 20%
(v/v) Knockout SR (Invitrogen, Carlsbad, CA). After the human iPSCs were trypsinized,
embryoid bodies (EBs) were generated by harvesting 9000 or 12,000 cells and seeding them
onto low-attachment 96-well plates (Lipidure Coat, NOF) in a differentiating medium, which
comprised DMEM/F-12 (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum, 1%
nonessential amino acids, 1% L-glutamine (Nacalai Tesque), 110 µM StemSure
2-mercaptoethanol solution (Wako), and 0.5% penicillin-streptomycin (Nacalai Tesque) with
10 µM Y27632 (Wako). Six days later, EBs were plated onto gelatin-coated tissue culture
24-well plates and grown for several weeks. The media were exchanged every 4 to 5 days
with the differentiating medium without Y27632.
solution: 0.25% (v/v) trypsin, 0.1 mg/mL collagenase IV, 1 mM CaCl2, and 20%
(v/v) Knockout SR (Invitrogen, Carlsbad, CA). After the human iPSCs were trypsinized,
embryoid bodies (EBs) were generated by harvesting 9000 or 12,000 cells and seeding them
onto low-attachment 96-well plates (Lipidure Coat, NOF) in a differentiating medium, which
comprised DMEM/F-12 (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum, 1%
nonessential amino acids, 1% L-glutamine (Nacalai Tesque), 110 µM StemSure
2-mercaptoethanol solution (Wako), and 0.5% penicillin-streptomycin (Nacalai Tesque) with
10 µM Y27632 (Wako). Six days later, EBs were plated onto gelatin-coated tissue culture
24-well plates and grown for several weeks. The media were exchanged every 4 to 5 days
with the differentiating medium without Y27632.
4
Sphere-Formation Assay for Cell Self-Renewal
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To evaluate the self-renewal activity of each cell line, a sphere-formation assay was performed using the protocol described in a previous report with a slight modification62 (link). Cells were seeded at 2000 cells/mL in an ultra-low attachment 24-well plate (Corning). These cells were cultured in serum-free DMEM/F12 (Nacalai Tesque) supplemented with bovine serum albumin (Nacalai Tesque), 5 mM HEPES (Nacalai Tesque), 100 U/mL penicillin (Nacalai Tesque), 100 µg/mL streptomycin (Nacalai Tesque), 20 ng/mL human recombinant EGF (Peprotech, NJ), 10 ng/mL human recombinant bFGF (Peprotech), and 1% B27 supplement (Invitrogen, CA). EGF, bFGF, and B27 supplement were used to stabilise the formation and maintenance of sphere. DMEM/F12 medium supplemented with 2 ng/mL EGF and 1 ng/mL bFGF was added to the culture every other day for 10 days. Spheres were counted under a light microscope at 40 × magnification. Each experiment was performed in triplicate and repeated at least thrice.
5
Quantifying Pancreatic Cancer Spheroid Formation
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Suspensions of PanIN and IPMN cells were adjusted to a density of 104 cells/ml in medium comprising DMEM/F12 (Nacalai, Japan) supplemented with B27 (Gibco, USA), 100 μg of epidermal growth factor (Thermo, USA), and 50 μg of basic fibroblast growth factor (Thermo, USA). Next, cells were seeded in semi-suspension and cultured on Ultra Low Attachment 6-well plates (Corning Incorporated Life Sciences, USA). After a 5 day incubation, the numbers of cell spheres were counted and recorded using cell3imager (SCREEN Holdings, Japan).
6
Cell Culture of RPE1 and HCT116 Cells
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RPE1 and HCT116 cells obtained from the American Type Culture Collection (ATCC) were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, Nacalai Tesque) and McCoy’s 5A medium (Thermo Fisher Scientific) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin, respectively. Cells were cultured at 37 °C in a humidified 5% CO2 incubator.
7
Inducing Ciliogenesis in hTERT-RPE1 Cells
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hTERT-RPE1 cells were cultured in DMEM/F-12 (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) and 0.348% sodium bicarbonate. To induce ciliogenesis, cells were grown to 100% confluence on coverslips and starved for 24 h in Opti-MEM containing 0.2% bovine serum albumin.
For immunofluorescence analysis, cells were fixed and permeabilized with 3% paraformaldehyde at 37°C for 5 min, and subsequently in 100% methanol for 5 min at −20°C, and washed three times with phosphate-buffered saline. The fixed/permeabilized cells were blocked with 10% FBS and stained with antibodies diluted in 5% FBS. The stained cells were observed using an Axiovert 200M microscope (Carl Zeiss). Statistical analyses were performed using JMP Pro 13 software (SAS Institute).
TIRF microscopy was performed as described previously (Takahashi et al., 2012 (link); Kubo et al., 2015 (link)). In short, RPE1 cells expressing EGFP-DYNC2LI1 were serum starved for 24 h on a glass-bottom culture dish, placed on a microscope stage prewarmed to 37°C, and observed using a TIRFM ECLIPSE Ti microscope (NIKON) at a video rate using NIS-Elements imaging software. Kymograms were generated with NIS-Elements imaging software.
For immunofluorescence analysis, cells were fixed and permeabilized with 3% paraformaldehyde at 37°C for 5 min, and subsequently in 100% methanol for 5 min at −20°C, and washed three times with phosphate-buffered saline. The fixed/permeabilized cells were blocked with 10% FBS and stained with antibodies diluted in 5% FBS. The stained cells were observed using an Axiovert 200M microscope (Carl Zeiss). Statistical analyses were performed using JMP Pro 13 software (SAS Institute).
TIRF microscopy was performed as described previously (Takahashi et al., 2012 (link); Kubo et al., 2015 (link)). In short, RPE1 cells expressing EGFP-DYNC2LI1 were serum starved for 24 h on a glass-bottom culture dish, placed on a microscope stage prewarmed to 37°C, and observed using a TIRFM ECLIPSE Ti microscope (NIKON) at a video rate using NIS-Elements imaging software. Kymograms were generated with NIS-Elements imaging software.
8
Isolation and Decidualization of Endometrial Cells
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Collected HESCs were isolated, cultured, and maintained as described previously20 (link),55 (link). We obtained the endometrial samples in Dulbecco’s modified Eagle’s medium-Ham’s (DMEM) F-12 (Nacalai Tesque, Kyoto, Japan) containing 1% (v/v) antibiotic solution, minced finely and digested enzymatically with 5 mg collagenase (5 mg/100 μl) (Sigma-Aldrich, Saint Louis, USA) and deoxyribonuclease (DNase) type I (100 μg/μl) (Roche Applied Science, Mannheim, Germany) for 1 h at 37 °C. After centrifugation, the cells were suspended in DMEM/F12 culture media containing 10% (v/v) dextran-coated charcoal (DCC) treated fetal bovine serum, 1% antibiotic solution, 1% (v/v) l -glutamine, 0.2% (v/v) insulin and 1 nM estradiol (all Sigma-Aldrich). Endometrial cells were cultured until confluence in 75 cm2 culture flasks at 37 °C in 5% carbon dioxide and then passaged once or twice. Cells were discarded after passage 3. In decidualization experiments, confluent monolayers were maintained in DMEM/F12 without phenol red (GIBCO, life technologies, Grand Island, USA) containing 2% (v/v) DCC-FBS and treated with 0.5 mM 8-bromo-cAMP, 1 μM P4 in combination with or without 100 µM resveratrol (all Sigma-Aldrich).
9
Maintenance of Undifferentiated hiPSC Lines
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The hiPSC lines 246H1 (a gift from Dr Yamanaka at Kyoto University, Kyoto, Japan) and TIG3/KOSM #7 were maintained as previously described37 (link). Undifferentiated hiPSCs were maintained on a feeder layer of mitomycin-treated SNL76/7 cells (DS Pharma Biomedical, Osaka, Japan) in DMEM/F12 (Nacalai Tesque, Kyoto, Japan) supplemented with 20% Knockout-Serum Replacement (Invitrogen, Carlsbad, CA, USA), 0.1 mM nonessential amino acids (Wako, Osaka, Japan), 0.1 mM β-mercaptoethanol, 50 U/mL penicillin and 50 mg/mL streptomycin (Nacalai Tesque), and 5 ng/mL bFGF (PeproTech, Rocky Hill, NJ, USA).
10
Inducing and Analyzing Cilia Formation
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The hTERT-RPE1 cells (CRL-4000; American Type Culture Collection) were cultured in DMEM/F-12 (Nacalai Tesque) supplemented with 10% FBS and 0.348% sodium bicarbonate. To induce ciliogenesis, cells were grown to 100% confluence on dishes or coverslips and starved for 24 h in Opti-MEM (Invitrogen) containing 0.2% bovine serum albumin. Expression vectors were transfected into the cells using X-tremeGENE9 DNA Transfection Reagent (Roche Applied Science).
Immunofluorescence analysis was performed as described previously (Takahashi et al., 2012 (link); Nozaki et al., 2017 (link)). Cells were fixed with 3% paraformaldehyde at 37°C for 15 min, washed three times with phosphate-buffered saline (PBS), quenched with 50 mM NH4Cl for 10 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 for 5 min, and washed three times with PBS. For the detection of endogenous RABL2, cells were fixed with 10% trichloroacetic acid on ice for 15 min. The fixed/permeabilized cells were blocked with 10% FBS and treated with antibodies diluted in 5% FBS. For the detection of γ-tubulin, antibodies were diluted with Can Get Signal immunostain (Toyobo). The stained cells were observed using an Axiovert 200 M microscope (Carl Zeiss) or an A1R-MP confocal laser-scanning microscope (Nikon).
Immunofluorescence analysis was performed as described previously (Takahashi et al., 2012 (link); Nozaki et al., 2017 (link)). Cells were fixed with 3% paraformaldehyde at 37°C for 15 min, washed three times with phosphate-buffered saline (PBS), quenched with 50 mM NH4Cl for 10 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 for 5 min, and washed three times with PBS. For the detection of endogenous RABL2, cells were fixed with 10% trichloroacetic acid on ice for 15 min. The fixed/permeabilized cells were blocked with 10% FBS and treated with antibodies diluted in 5% FBS. For the detection of γ-tubulin, antibodies were diluted with Can Get Signal immunostain (Toyobo). The stained cells were observed using an Axiovert 200 M microscope (Carl Zeiss) or an A1R-MP confocal laser-scanning microscope (Nikon).
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