Tested and authenticated CAL27 cells were purchased from ATCC. HSC-3 cells were a kind gift from Roberto Weigert (NIH, Bethesda, MD), and were described previously [11 ]. CAL27 and HSC-3 cells were cultured under standard conditions in DMEM (Mediatech, Herndon, VA) supplemented with 10% FBS (Sigma-Aldrich). Fibroblast culture was established from human tongue OSCC specimens (see Human oral tissue specimens). Biopsy was cut into small pieces and dissociated enzymatically by 0.25% collagenase (Worthington Biochemical Corporation, Lakewood, NJ) in DMEM with 20% fetal bovine serum. Digested tissue was placed in a culture dish in 5 ml of DMEM with 20% fetal bovine serum and grown for 3–5 days. The resulting confluent culture was subsequently passaged in DMEM with 10% fetal bovine serum.
Dulbecco modified eagle medium (dmem)
Manufactured by Worthington
Sourced in United States
DMEM (Dulbecco's Modified Eagle Medium) is a commonly used cell culture media that provides nutrients and growth factors essential for the maintenance and proliferation of various cell types in vitro. It is a complex, buffered medium that supports the growth of a wide range of cell lines.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Collagenase, by Worthington (2 mentions)
Neutral protease, by Worthington (2 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Normocin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Apc anti mouse cd45, by BD (1 mentions)
7 aad, by BD (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Bovine serum albumin and trypsin inhibitor, by Worthington (1 mentions)
Percp cy5.5 conjugated anti cd45 antibody, by BioLegend (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Collagenase type 4, by Worthington (1 mentions)
Collagenase 2, by Worthington (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Trypsin edta, by Fujifilm (1 mentions)
Mito serum extender, by BD (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
12 protocols using dulbecco modified eagle medium (dmem)
1
Establishing Oral Cancer Cell Lines
2
Isolation and Culture of Rat MSCs and Cardiomyocytes
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A total of 90 healthy male Sprague Dawley (SD) rats (age: 7 – 8 weeks, weight: 250 – 280 g) (Guangdong Medical Laboratory Animal Center, Foshan, Guangdong, China) were housed at 20 - 25°C in a constantly humidified atmosphere under a 12-h light/dark cycle with free access to water and food. The animal treatment was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Guangxi International Zhuang Medical Hospital Affiliated to Guangxi National University of Traditional Chinese Medicine.
MSCs were isolated from healthy 8-week SD rats with reference to the methods reported previously [33 (link), 34 (link)] and cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (streptomycin and penicillin).
Neonatal cardiomyocytes were isolated from 2-day newborn rats using the Neonatal Cardiomyocytes Isolation kit (Worthington Biochemical Corp., Lakewood, NJ, USA) and cultured in DMEM (Hyclone, Logan, Utah, USA) containing 10% FBS.
MSCs were isolated from healthy 8-week SD rats with reference to the methods reported previously [33 (link), 34 (link)] and cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (streptomycin and penicillin).
Neonatal cardiomyocytes were isolated from 2-day newborn rats using the Neonatal Cardiomyocytes Isolation kit (Worthington Biochemical Corp., Lakewood, NJ, USA) and cultured in DMEM (Hyclone, Logan, Utah, USA) containing 10% FBS.
3
Isolation and Culture of Chondrocytes from Cartilage
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The collection of cartilage was approved by the Ethics Committee of Shenzhen Second People's Hospital. Samples were collected after obtaining written informed consent from all individuals. All the cartilage samples were obtained from volunteer donors (26–35 years old, male) after trauma patients, in a physiological saline system containing penicillin/streptomycin (P/S), and were processed within 6 hr of collection. For isolation of chondrocytes, the cartilage specimens were minced to 1 mm3 and digested in 1 mg/mL type II collagenase in Dulbecco's modified Eagle's medium (DMEM; Worthington Biochemical Corporation, USA) for 8 hr at 37°C in a shaker. After filtration, cells were harvested and seeded onto tissue culture flasks at a density of 1 × 104 cells/cm2 and subcultured in chondrocyte growth medium (DMEM-F12, 10% FBS, 10 μg/L bFGF, and 0.1 mg/mL P/S). All incubations occurred in a 5% CO2 atmosphere at 37°C. Medium was replaced 3 times a week until cells reached confluence. At 80% confluence, cells were harvested with a trypsin/EDTA solution (Gibco/Life Technologies, Australia) and seeded onto new flasks. Chondrocytes of passage 2 (P2) were used for chondrocyte induction [27 (link)].
4
Dorsal Root Ganglion Dissociation Protocol
5
Isolation and Characterization of Stromal Vascular Fraction from Adipose Tissue
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Epididymal adipose tissue was excised from Prkaa1ΔMφ and Prkaa1WT mice and minced into small pieces, digested with collagenase type I (5 mg/ml, Cat. No. 4194, Worthington, Lakewood, NJ, United States) in DMEM (Cat. No. 11054020, Gibco, Waltham, MA, United States) for 30 min at 37°C with shaking. After centrifugation at 500 g for 5 min, the stromal vascular fraction (SVF) was resuspended in FACS buffer (0.5% FBS in PBS) and passed through a 70-μm cell strainer. The cells were incubated with FcR blocking reagent (Cat. No. 553142, BD Biosciences, San Jose, CA, United States) for 10 min at 4°C, then incubated with 7-AAD (Cat. No. 559925, BD Biosciences, San Jose, CA, United States), PE anti-mouse CD31 (4 μg/ml; Cat. No. 553373, BD Biosciences, San Jose, CA, United States) and APC anti-mouse CD45 (3 μg/ml; Cat. No. 558702, BD Biosciences, San Jose, CA, United States) for 30 min at 4°C. After washing, the cells were sorted using a FACS Caliber. The CD45+CD31–7-AAD– population was collected for RT-PCR analysis.
6
Quantifying Collagen Crosslinks in Tissue
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Tissue samples (M-side and L-side sets) from 9 patients (1 female, 8 males; mean age 52.88 months, SD = 21.64) were subjected to a defined collagenase treatment (300 U/mL of clostridial collagenase type III, dissolved in DMEM; Worthington Biochemical Corp., Lakewood, NJ, USA), 250 µL of enzyme solution per 10 mg of tissue. The samples were incubated at 37 °C with mild vortexing until they completely degraded into small pieces. After collagenase digestion treatment, the samples were lyophilized and were weighed. The samples were then proteolytically digested using acid hydrolysis (50 µL of 6N HCl, 105 °C overnight) and were neutralized (50 µL of 6M NaOH). The concentration of pyridinoline and deoxypyridinoline crosslinks was measured by ELISA, using the MicroVue Serum PYD Enzyme Immunoassay kit (Cat. No. 8019, MicroVue, Quidel, San Diego, CA, USA). The concentration of crosslinks was normalized to the collagen content of the same sample measured in parallel by a hydroxyproline colorimetric assay (K555, BioVision, Milpitas, CA, USA), according to the manufacturer’s instructions.
7
Isolation of Primary Chondrocytes from Knee Tissue
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The discarded surgical tissue of patients who had undergone total knee replacement was collected (n = 5). Macroscopically normal cartilage tissue was dissected from the joint under sterile conditions and minced into pieces measuring <1 mm3. These pieces were then sequentially digested, first with hyaluronidase (30 min, 660 U/ml in DMEM; Sigma) and then with collagenase (overnight; 583 U/ml in DMEM with 10% FBS; Worthington Biochemical). This resulted in a cell slurry which was washed through a 70-μm filter with DMEM, centrifuged (600 RCF, 5 min) and plated (6000 cells/cm2) into tissue culture flasks in growth media (DMEM supplemented with 10% FBS 1% pen/strep) from which the primary cell culture (p0) cell stocks were produced. Once the tissue culture flasks reached 90% confluence, they were trypsinized and cryogenically stored (95% FBS, 5% DMSO).
8
Nociceptive Primary Cell Culture
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Dorsal root ganglia L4 – L6 were used as the source for a nociceptive primary cell culture population. The DRG tissues were dissected and collected immediately post mortem27 (link). The collected samples were initially dissociated using a solution of DMEM, neutral protease, and collagenase (Worthington Biochemical) at a temperature of 37 °C on a rocker at a low setting for 40 minutes. The tissue was further dissociated by mechanical trituration and plated on poly-L lysine and laminin coated coverslips (BD Biosciences) in a culture media of 1 mg/mL bovine serum albumin and trypsin inhibitor (Worthington Biochemical). Following plating, cells were incubated for 2–3 hours, and then another 1 mL of culture media was adding to each well. The cells were then incubated overnight for 12–15 hours at a temperature of 37 °C with 5% CO2 to improve adhesion to coverslips prior to experimentation. Some sensory neurons were exposed to FITC-TAT-4BB for 15 minutes using 15 μM solution.
9
Isolation and Culture of Murine Synovial Fibroblasts
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SFs were isolated from the paws of mice after one/two MSU-or zymosan-injections, and from noninjected naïve paws. SF cell suspensions of murine paws were prepared by gently rocking dissected and skin-flayed paws for 1 hour at 37°C in DMEM containing 1 mg/ml collagenase type IV (Worthington Biochemicals, #LS004188). Cells derived from 2 to 3 paws were pooled and cells were then grown in DMEM (Gibco) supplemented with 10% heat-inactivated calf serum, (glucose (4.5 g/L), L-glutamine 2 mM), penicillin and streptomycin (1% each) antibiotics, and 1% fungizone (Amphotericin B, Sigma Aldrich). Medium was changed twice a week. SFs between passages 4 and 8 were used for the experiments and contained less than 2% CD45 + or CD31 + cells, as determined by flow cytometry with a PerCP Cy5.5 conjugated anti-CD45 antibody (Biolegend, #103132) and an AF647-conjugated anti-CD31 antibody (Biolegend, #102516). Alternatively, we used flow cytometry sorted CD45 -CD31 -SFs.
10
Isolation of Aortic Vascular Smooth Muscle Cells
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Cryopreserved heterozygous LPP knockout (LPP-KO) embryos on a mixed 129/SvJ-C57BL/6J background were kindly provided by Professor Wim Van de Ven at the Department of Human Genetics, KU Leuven, Belgium. They were transferred to pseudopregnant C57BL6/J foster mothers, and the resulting offspring were backcrossed onto the C57BL6/J background at least ten times. Wildtype (WT) mice referred to herein represent littermates derived from the breeding of heterozygous LPP-KO mice.
For preparing VSMCs from these mice, the descending aorta starting at the outlet of the right renal artery was dissected and washed twice in calcium-free Dulbecco’s PBS. The aorta was then briefly (15 min) pre-digested in collagenase II-containing DMEM (1 mg/mL, Worthington Biochemical, Lakewood, NJ, USA) to easily peel off the adventitial layer. Subsequently, the aorta was cut in 1 mm rings and digested in collagenase II (1 mg/mL) and elastase (0.15 mg/mL) for approximately 3 h, followed by cell dispersion. VSMCs were cultured in DMEM supplemented with 15% foetal bovine serum (FBS) plus penicillin/streptomycin and fungizone at 37 °C with 5% CO2. Cells in passages 3–5 were used in experiments.
For preparing VSMCs from these mice, the descending aorta starting at the outlet of the right renal artery was dissected and washed twice in calcium-free Dulbecco’s PBS. The aorta was then briefly (15 min) pre-digested in collagenase II-containing DMEM (1 mg/mL, Worthington Biochemical, Lakewood, NJ, USA) to easily peel off the adventitial layer. Subsequently, the aorta was cut in 1 mm rings and digested in collagenase II (1 mg/mL) and elastase (0.15 mg/mL) for approximately 3 h, followed by cell dispersion. VSMCs were cultured in DMEM supplemented with 15% foetal bovine serum (FBS) plus penicillin/streptomycin and fungizone at 37 °C with 5% CO2. Cells in passages 3–5 were used in experiments.
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