NIH-3T3 (ATCC, USA) cells were cultured in a T75 flask (NUNC, USA) for 2 days with Dulbecco's modified Eagle's medium (DMEM, Welgene Inc., Republic of Korea) containing antibiotics (Welgene Inc., Republic of Korea) and 10% fetal bovine serum (FBS, Welgene Inc., Republic of Korea). NIH-3T3 cells were seeded at a density of 2 × 106 cells mL-1. DPSCs (Intellectual Biointerface Engineering Center, Dental Research Institute, Seoul National University, SNUDH_IRB No. CRI05004) were cultured in a 90 mm dish (NUNC, USA) for 2 days with α-minimum essential medium (α –MEM, Welgene Inc., Republic of Korea) containing 10% fetal bovine serum (FBS, Welgene Inc., Republic of Korea) and antibiotics (Welgene Inc., Republic of Korea). DPSCs were seeded at a density of 1 × 106 cells mL-1. All cells were cultured at 37 °C and 5% CO2 in an incubator (Steri-Cycle 370 Incubator, Thermo Fisher Scientific, USA).
Steri cycle 370 incubator
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Steri-Cycle 370 Incubator is a laboratory equipment designed for incubation applications. It provides a controlled environment for various experimental and testing procedures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Welgene (6 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (3 mentions)
L ascorbic acid, by Thermo Fisher Scientific (2 mentions)
T75 flask, by Thermo Fisher Scientific (1 mentions)
α mem, by Welgene (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Ls 015 10, by Welgene (1 mentions)
Sodium bicarbonate, by Duksan Pure Chemicals (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Infinite 200 pro m nano, by Tecan (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
9 protocols using steri cycle 370 incubator
1
Cell Culture Protocols for NIH-3T3 and DPSCs
2
Osteogenic Differentiation of hABMSCs
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hABMSCs were collected at the Intellectual Biointerface Engineering Center, Dental Research Institute, College of Dentistry, Seoul National University. hABMSCs were placed in 35 mm culture dishes at a density of 1.0 × 104 cells/cm2 and cultured for 5 and 10 days. Cells were cultured in α-minimum essential medium (α-MEM) containing 10% fetal bovine serum (FBS, Welgene Inc., Republic of Korea) and 10 mM ascorbic acid (L-ascorbic acid) and antibiotics (Antibiotic-Antimycotic solution, Gibco) at 37°C in a humidified atmosphere of 5% CO2 (Steri-Cycle 370 Incubator, Thermo Fisher Scientific, USA). The cells were then incubated with osteogenic medium (100 nM dexamethasone, 50 μg/mL of ascorbic acid, and 10 nM of β-glycerophosphate; Sigma) for 10 days. The induction culture medium was changed every second or third day. The proliferation and osteogenic differentiation of the cells were examined after exposure to each OSS.
3
Dental Pulp Stem Cell Isolation and Culture
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The DPSCs were collected at the Intellectual Biointerface Engineering Center, Dental Research Institute, College of Dentistry, Seoul National University. All experiments related to the DPSCs were approved by the Seoul National University Institutional Animal Care and Use Committee (SNU-120427-2-2). The cells were cultured in α-minimum essential medium (MEM) containing 10% fetal bovine serum (FBS, Welgene Inc., Gyeongsan, Korea), 10 mM ascorbic acid (l -ascorbic acid, Simga-Aldrich, St. Louis, MO, USA), antibiotics (Welgene Inc., Gyeongsan, Korea), and sodium bicarbonate (DUKSAN, Seoul, Korea) at 37 °C in a humidified atmosphere of 5% CO2 (Steri-Cycle 370 Incubator, Thermo Fisher Scientific, Waltham, MA, USA). The medium was changed every other day. When the cells became confluent, they were detached with 1 mL trypsin-EDTA solution (LS 015-10, Welgene Inc., Gyeongsan, Korea), counted, and passaged.
4
Osteogenic Differentiation of hBMSCs
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The hBMSCs were received from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and cultured as previously reported [22 ,23 (link)]. The cell culture was carried out using DMEM supplemented with 10% FBS and 1% antibiotics containing penicillin (10,000 units/mL), streptomycin (10,000 μg/mL), and amphotericin B (25 μg/mL) at 37°C in a humidified atmosphere of 5% CO2 (Steri-Cycle 370 Incubator; Thermo Fisher Scientific, USA). The old media were changed with fresh media every three days during the experiment. After 70–80% confluency, the hBMSCs were treated with different concentrations of CPI for the desired periods. Passage 5 cells were used in this study. For osteogenic induction, the cells were cultured in an osteogenic induction media containing DMEM supplemented with 50 μg/mL L-ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone.
5
Culturing Human Adipose-Derived Mesenchymal Stem Cells
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The hABMSCs were received from the Korean Cell Line Bank (KCLB; Seoul National University, Korea), and cell culture was performed according to the method described by Gronthos et al. [24 (link)]. Briefly, the cells were cultured in α-minimum essential media (MEM) containing 10% fetal bovine serum (FBS, Welgene Inc., Gyeongsan, Korea), 10 mM ascorbic acid (L-ascorbic acid), 1% antibiotics (Anti-Anti, 100×, Gibco-BRL, Gaithersburg, MD, USA), and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified atmosphere with 5% CO2 (Steri-Cycle 370 Incubator, Thermo Fisher Scientific, USA) for desired periods. Passage three hABMSCs were used for in vitro experiments.
6
Culturing and Passaging Human Bone Marrow Stromal Cells
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The hBMSCs were received from the Korean Cell Line Bank (KCLB) (Seoul National University), Republic of Korea. The cells were cultured as previously reported somewhere else.23 In brief, the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Welgene Inc., Republic of Korea) having 10% fetal bovine serum (FBS) (Welgene Inc., Republic of Korea), and 1% antibiotic (Anti–Anti; 100×, Gibco-BRL, USA) at 37 °C in a humidified atmosphere containing 5% CO2 (Steri-Cycle 370 Incubator; Thermo-Fischer Scientific, USA). The old media were replaced with fresh media after three days. At ∼70–80% of confluency, the cells were detached, counted, and passaged with 1 mL of 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) (Gibco, USA) solution. Passage three were used for the primary cell culture.
7
Osteogenic Differentiation of hBMSCs
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The hBMSCs were received from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and cultured as previously reported. Briefly, the cell culture was carried out using DMEM supplemented with 10% FBS and 1% antibiotics containing penicillin (10,000 units/mL), streptomycin (10,000 µg/mL), and amphotericin B (25 µg/mL) at 37 °C in a humidified atmosphere of 5% CO2 (Steri-Cycle 370 Incubator; Thermo Fisher Scientific, Waltham, MA, USA). After 70–80% confluency, the hBMSCs were treated with different concentrations of PPI for the desired periods. Passage 5 cells were used in this study. For the osteogenic induction, the cells were cultured in an osteogenic induction media containing DMEM supplemented with 50 µg/mL L-ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone.
8
Cytotoxicity Evaluation of Mesenchymal Stem Cells
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The human mesenchymal stem cells (hMSCs) derived from the bone (Korean Cell Line Bank, Republic of Korea) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Welgene, Seoul, Republic of Korea) with 10% fetal bovine serum (Welgene, Seoul, Republic of Korea) and 1% antibiotics (Welgene, Seoul, Republic of Korea). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 (Steri-Cycle 370 Incubator, Thermo Fischer Scientific, USA) environment. The old culture media were replaced with fresh media every 3 days' time intervals during the experiment. After ∼80–90% of cell confluency, the cells were detached with 1 mL trypsin–ethylene diamine tetra acetic acid (EDTA, Gibco, Waltham, Massachusetts, USA) solution followed by the rinsing with phosphate-buffered saline (PBS). The cells were used between three and five passages.
The fabricated scaffolds were placed in a 24-well plate and were incubated at 37 °C in 5% CO2 for different time periods. Cytotoxicity of the fabricated PC and GPC scaffolds was evaluated through WST-1 assay. For this, the cultured cells were treated with 100 μL of WST-1 reagent to produce the formazan dye which was quantified with the spectrometer (Infinite® M Nano 200 Pro, TECAN, Switzerland) by taking the absorbance at 490 nm. The samples were taken in the triplicate fashion, and data are given as mean ODs ± standard deviations.
The fabricated scaffolds were placed in a 24-well plate and were incubated at 37 °C in 5% CO2 for different time periods. Cytotoxicity of the fabricated PC and GPC scaffolds was evaluated through WST-1 assay. For this, the cultured cells were treated with 100 μL of WST-1 reagent to produce the formazan dye which was quantified with the spectrometer (Infinite® M Nano 200 Pro, TECAN, Switzerland) by taking the absorbance at 490 nm. The samples were taken in the triplicate fashion, and data are given as mean ODs ± standard deviations.
9
Isolation and Culture of PDL Cells
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As described previously, the primary culture of PDL cells was conducted via the direct cell outgrowth method by isolating cells from the PDL tissue around the middle third of extracted healthy human teeth [129 (link),130 (link)]. Cells were maintained in Minimum Eagle Medium-α (MEM-α) (Gibco, USA) augmented with 10% fetal bovine serum (FBS) (Gibco, USA), 1% penicillin/streptomycin (P/S), and 1% amphotericin B (Gibco, USA) in a Steri Cycle 370 incubator (Thermo-Fisher Scientific, USA) with a humid atmosphere containing 95% air and 5% CO2 at 37°C. Cell outgrowths were passaged before reaching confluency. Only cells passaged three to seven were used for experimentation. Cells were validated by morphological assessment and gene expression of confident biomarkers such as Periostin.
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