A total of 6 samples were prepared. Cells were seeded into 6-well plates and pre-cultured for 12 h for adhesion. Then, three cell samples were moved to a tri-gas incubator (Thermo Fisher Scientific, Waltham, MA, USA), and culturing was continued at 37 °C with 1% O2 and 5% CO2 for another 24 h. The other three cell samples were set as control groups and were cultured at 37 °C with 20% O2 and 5% CO2 for 24 h. Total protein extraction was then performed using a lysis solution followed by subsequent ultrasonication and centrifugation. The supernatant was collected, and the concentration was determined by the BCA method. The samples were then stored at − 80 °C for isobaric tags for relative and absolute quantitation analysis.
Tri gas incubator
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Tri-gas incubator is a laboratory equipment designed to provide a controlled environment for cell culture applications. It maintains consistent temperature, humidity, and gas composition (carbon dioxide, oxygen, and nitrogen) within the incubation chamber.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Roswell park memorial institute (rpmi), by Merck Group (2 mentions)
A769662, by R&D Systems (2 mentions)
Compound c, by R&D Systems (2 mentions)
Recombinant human tgf β, by R&D Systems (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Streptomycin, by Beyotime (2 mentions)
Penicillin, by Beyotime (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Gelatin, by Merck Group (1 mentions)
Tca8113, by National Collection of Authenticated Cell Cultures (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Scc15, by National Collection of Authenticated Cell Cultures (1 mentions)
Mouse recombinant leptin, by Thermo Fisher Scientific (1 mentions)
Methacrylic anhydride, by Merck Group (1 mentions)
Omnicure s2000 uv lamp, by Excelitas (1 mentions)
Aml12, by Thermo Fisher Scientific (1 mentions)
30 protocols using tri gas incubator
1
Protein Extraction and Quantification
2
Astrocyte Hypoxia Response Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, following a wash with glucose-free culture medium, primary astrocyte cultures were supplied with fresh glucose-free medium in the absence of serum. For the hypoxic conditions, the cells were placed in a humidified Tri-gas incubator (Thermo Scientific, Waltham, MA) for 24 hours, with conditions of 94% N2/5% CO2/1% O2. Following the 24-hour incubation, the cells were returned to standard incubator conditions (see Section 2.1 ) for an additional 24 hours. Control cells, used in all assays, were maintained in normal incubator conditions and fed with glucose-containing DMEM.
3
Isolation and Hypoxic Culture of Rat Cardiac Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hearts were excised from 2-day-old neonatal SD rats and then minced into 0.5–1-mm3 patches with sterile scissors. The pieces were digested with 1 mg/ml collagenase II for 15 min at 37 °C three times in PBS. The cells collected were seeded in 60-mm plates coated with gelatin (g5384, Sigma) in DF12 medium with 10% FBS. The un-adhered cells were transferred onto another plate coated with matrigel (Corning, 356,231) after 1 h. On the next day, the culture medium was switched, and beating myocardial cells (CMs) were observed 24 h later. Apart from the un-adhered cells, most of the adhered cells were fibroblasts, and after passaging two to three times with DF12 + 10%FBS, the spindle-shaped cells were fibroblasts (HFs).
For hypoxic culture, cells (CMs, HFs, and MSCs) were cultured in a tri-gas incubator (Thermo Fisher Scientific, Marietta, OH, USA) composed of 94% N2, 5% CO2, and 1% O2 with the same culture medium (DF12 and 10% FBS) for 24 h. Cells were then harvested for RT-qPCR analysis of IL-33 levels.
For hypoxic culture, cells (CMs, HFs, and MSCs) were cultured in a tri-gas incubator (Thermo Fisher Scientific, Marietta, OH, USA) composed of 94% N2, 5% CO2, and 1% O2 with the same culture medium (DF12 and 10% FBS) for 24 h. Cells were then harvested for RT-qPCR analysis of IL-33 levels.
4
Hypoxia-induced miR-671-5p and circCDR1as
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines Tca-8113, SCC-15, and HOK were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells that were cultured in RPMI-1640 (RPMI 1640, Gibco, USA) or Dulbecco’s modified Eagle’s medium (HyClone, USA) contained 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C in a humidified 5% CO2 atmosphere. Hypoxia treatment was performed using a tri‑gas incubator (Thermo, MA, USA) consisting of 5% CO2, 93% N2, and 2% O2 for different periods (4, 8, 16, and 24 h).
MiR-671-5p mimics or Negative Control were designed and synthesized by GenePharma. The sequence of miR-671-5p mimics was 5′-AGGAAGCCCUGGAGGGGCUGGAG-3′. Cells were transfected with the oligonucleotides using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s instructions. CircCDR1as-coding sequence was cloned into pLCDH-ciR vector (Geenseed Biotech, Guangzhou, China). Then, circCDR1as stable overexpression cell lines were constructed following the manufacturer’s instructions. The efficiency of circCDR1as overexpression was confirmed by qRT-qPCR.
MiR-671-5p mimics or Negative Control were designed and synthesized by GenePharma. The sequence of miR-671-5p mimics was 5′-AGGAAGCCCUGGAGGGGCUGGAG-3′. Cells were transfected with the oligonucleotides using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s instructions. CircCDR1as-coding sequence was cloned into pLCDH-ciR vector (Geenseed Biotech, Guangzhou, China). Then, circCDR1as stable overexpression cell lines were constructed following the manufacturer’s instructions. The efficiency of circCDR1as overexpression was confirmed by qRT-qPCR.
5
Cell Culture Conditions and Treatments
6
BV2 Microglial Cells in Normoxia/Hypoxia with Leptin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The BV2 microglial cell line was purchased from ScienCell (CA, USA) and cultured in MEM medium with 10% FBS and 1% penicillin/streptomycin. For normoxia culture, cells were maintained in a humidified incubator containing 95% air and 5% CO2 at 37°C; for hypoxia culture, cells were maintained in a tri-gas incubator (Thermo Fisher Scientific, USA) with oxygen concentration at 1%. BV2 cells were cultured in the normoxia/hypoxia condition with/without the treatment of mouse recombinant leptin (Peprotech, UK) at the concentration of 1 μM for 24 h.
7
Photocrosslinkable Hydrogel for 3D Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Photocrosslinkable Me-HA (HA, ∼1200 kDa, NovaMatrix) and Me-Gel (type B gelatin, Sigma) were synthesized as previously reported [62 (link)] through the reaction of methacrylic anhydride (Sigma) with HA (0.5%) and gelatin (10%) solution. A hydrogel precursor solution composed with Me-HA (0.75%w/v) /Me-Gel (0.75%w/v) was dissolved in cell culture medium with 0.05% w/v 2-hydroxy-1(4-(hydroxyethyl)phenyl)-2-methyl-1-propanone (Irgacure 2959, CIBA Chemicals). The gel precursor was transferred into silicone molds (8 mm in diameter, 1mm in thickness) and subsequently exposed to OmniCure S2000 UV lamp (Lumen Dynamics) for 45 s at room temperature to generate hydrogel matrix. For breast cancer cell encapsulations into hydrogels, 21PT or 21MT-2 cell lines were used at a density of 5×106 cells/ml (Figure 1A ). These cell-laden hydrogels scaffolds were maintained in the cell culture medium at 37°C in either a normoxic (5% CO2, 21% O2) or hypoxic (5% CO2, 5% O2) environment for 14 days in trigas incubator (Thermo Fisher Scientific). The medium was replaced on alternate days.
8
Hypoxic Regulation of Liver Cell Crosstalk
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AML12 (alpha mouse liver 12 cells) and HSC-T6 (rat hepatic stellate cells) were purchased from the Shanghai Institute of Biochemistry and Cell Biology. Cells were cultured in DMEM-F12 (Hyclone, USA) supplemented with 10% heat-inactivated fetal bovine serum (Bioind, Israel), and were maintained in a humidified atmosphere with 5% CO2 at 37°C. For hypoxic exposure, AML12 cells were incubated in a tri-gas incubator (Thermo, America) of 1% O2, 5% CO2, and 94% N2. Serum-free “conditioned medium” (CM) was obtained from the supernatant of AML12 cells to eliminate the influence of serum cytokines. HSC-T6 cells were cultured in CM and the cells cultured in DMEM were used as control.
9
Lung Cancer Cells Hypoxia and Epigenetic Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human lung adenocarcinoma cell line, A549, was purchased from ATCC and cultured in DMEM medium (Gibco, Carlsbad, CA, U.S.A.) with 10% FBS (Gibco) at 37°C with 5% CO2. For hypoxia treatment, cells were cultured in tri-gas incubator (Thermo, MA, U.S.A.) consisting of 2% O2, 5% CO2 and 93% N2 for 24 h. Then, the cells were treated with the DNA methylation inhibitor 5-Aza-2′-deoxycytidine (Aza) (10 μM, Sigma, U.S.A.).
10
In Vitro Hypoxia Model for Myocardial Ischemia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro hypoxia cell models were established to simulate myocardial ischemia and infarction in vivo. The hypoxic solution was prepared according Koyama et al. [25 (link)]. Cardiomyocytes were incubated with hypoxic solution. The hypoxia models were prepared in a tri-gas incubator (Thermo, USA) with 95% N2 + 5% CO2 at 37 °C for 6 h.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!