Hypoxic chamber

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Hypoxic Chamber is a laboratory equipment designed to precisely control and maintain a low-oxygen, hypoxic environment. It allows researchers to simulate and study the effects of reduced oxygen levels on biological samples or cellular systems.

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31 protocols using hypoxic chamber

Ischemia-Reperfusion Injury in Neuroblastoma Cells

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Human neuroblastoma cells lines (SH-SY5Y) was a gift from Dr. Zan Huang (College of life sciences, Wuhan University). Cells were cultured in DMEM media (Hyclone South Logan, UT) supplemented with 10% FBS (Gibco, Grand Island, NY) and 1% penicillin-streptomycin (Hyclone, South Logan, UT) (named as cell growth media) at normal culture condition.
After the cell reached full confluence in the plate, in vitro ischemia and reperfusion was conducted. The culture media was replaced by DMEM medium (Gibco, Grand Island, NY) without glucose and FBS, and then settled immediately in hypoxic chamber (Thermo scientific, Marietta, OH) with the condition of 1% O₂, 5% CO₂ at 37°C, which is regard as ischemia [21] (link). After 2 hours, the cells were re-cultured in cell growth media. And another 12 hours (reperfusion) later, the cells were collected.

Tian H., Wang L., Cai R., Zheng L, & Guo L. (2014). Identification of Protein Network Alterations upon Retinal Ischemia-Reperfusion Injury by Quantitative Proteomics Using a Rattus norvegicus Model. PLoS ONE, 9(12), e116453.

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Neuroprotective Effects of Compound E

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The HT22 cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C. For oxygen-glucose deprivation (OGD) insult, the neurons were maintained in a hypoxic chamber (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 94% N₂, 1% O₂, and 5% CO₂ and then cultured in glucose-free DMEM solution at 37 °C for 0, 2, 4, 6, or 8 h, as previously described (Lai et al. 2020 ). After OGD, the neurons were cultured in normal DMEM solution with 10% FBS for 24 h. In the CE (MedChemExpress, NJ, USA) treatment groups, HT22 cells were pretreated with CE at doses of 1, 2, 4, or 8 µg/mL for 4 h prior to OGD/R (Tian et al. 2019 (link); Wang et al. 2020a (link)).

Li J., Bu Y., Li B., Zhang H., Guo J., Hu J, & Zhang Y. (2022). Calenduloside E alleviates cerebral ischemia/reperfusion injury by preserving mitochondrial function. Journal of Molecular Histology, 53(4), 713-727.

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In vitro Oxygen-Glucose Deprivation/Reoxygenation Model

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OGD/R was performed to generate I/R models in vitro. BV2 cells were seeded into 95-cm cell culture dishes at a density of 1×10⁶ cells/well and incubated at 37°C for 6 h. To mimic OGD/R injury, cells in the logarithmic growth phase were cultured in glucose-free DMEM and placed in a hypoxic chamber (Thermo Fisher Scientific, Inc.) supplemented with a gas mixture of 1% O₂, 94% N₂ and 5% CO₂ at 37°C for 6 h. OGD was terminated by restoration with high-glucose DMEM and incubation under normoxic conditions (95% air and 5% CO₂) at 37°C for 12 h. BV2 cells without any treatment were used as a control.

Hu W, & Lin C. (2020). S100a8 silencing attenuates inflammation, oxidative stress and apoptosis in BV2 cells induced by oxygen-glucose deprivation and reoxygenation by upregulating GAB1 expression. Molecular Medicine Reports, 23(1), 64.

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Hypoxic Stress on Cardiomyocytes

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Human cardiomyocyte line AC16 (Davidson et al., 2005 (link)) was got from American Type Culture Collection (Manassas, VA, USA) and fostered in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing nutrient mixture F-12 (Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), and 1% penicillin/streptomycin (Sigma-Aldrich). AC16 cells in the hypoxia group were incubated for the indicated time (24, 48, and 72 h) in a hypoxic chamber (Thermo Fisher Scientific, Inc.) filling with a gaseous mixture of 94% N₂, 5% CO₂, and 1% O₂. AC16 cells incubated in a normoxic environment (74% N₂, 5% CO₂, and 21% O₂) at 37°C were served as the normal group.

Zheng J., Peng B., Zhang Y., Ai F, & Hu X. (2020). FOXD3-AS1 Knockdown Suppresses Hypoxia-Induced Cardiomyocyte Injury by Increasing Cell Survival and Inhibiting Apoptosis via Upregulating Cardioprotective Molecule miR-150-5p In Vitro. Frontiers in Pharmacology, 11, 1284.

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Hypoxia-Induced Responses in A549 Cells

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Human pulmonary epithelial A549 cells were maintained in DMEM containing 10% FBS and penicillin/streptomycin. Cells were grown at 37°C in a humidified atmosphere of 95% air/5% CO₂ and fed every 2–3 days. Before treatment, the cells were washed with phosphate-buffered saline and cultured in DMEM/5% charcoal–dextran stripped FBS (CD-FBS) for 2 days. All treatments were done with DMEM/5% CD-FBS. For the hypoxic condition, cells were incubated with a mixture of 1% O₂, 5% CO₂, and 94% N₂ using a hypoxic chamber (Thermo Fisher Scientific, Waltham, MA, USA).

Lim W, & Kang C. (2020). Avenanthramide C suppresses hypoxia-induced cyclooxygenase-2 expression through sirtuin1 activation in non-small-cell lung cancer cells. Animal Cells and Systems, 24(2), 79-83.

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Hypoxia-Induced Necroptosis and Apoptosis

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The rat cardiomyocyte-derived H9c2 cell line (American Type Culture Collection) was cultured in high glucose-DMEM (GIBCO, Waltham, MA, USA) containing 10% FBS (Atlas Biologicals, Fort Collins, CO, USA) and 1% antibiotics (GIBCO). To induce necroptosis and apoptosis, cells were incubated in a hypoxic chamber (Thermo Fisher Scientific) with 1% O₂, 5% CO₂, and 94% N₂. After exposure to hypoxia, cell counting kit solution (CCK-8, Dogen, Seoul, Korea) was added to each well at a final concentration of 0.5 mg/mL and incubated for 2 h at 37°C. Cell viability was determined by measuring cells at 450 nm.

Shin S., Choi J.W., Moon H., Lee C.Y., Park J.H., Lee J., Seo H.H., Han G., Lim S., Lee S., Kim S.W, & Hwang K.C. (2019). Simultaneous Suppression of Multiple Programmed Cell Death Pathways by miRNA-105 in Cardiac Ischemic Injury. Molecular Therapy. Nucleic Acids, 14, 438-449.

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Maintenance and Hypoxic Cultivation of Lung and Breast Cell Lines

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Maintenance of human lung epithelial A549 and human breast cancer MCF-7 cells as described previously [30] (link). AR-EcoScreen™ cells (gift from Dr, Mitsuru Iida, Hiyoshi Corporation, Japan) were derived from a Chinese Hamster Ovary cell line that is stably transformed with a plasmid containing an androgen response element (ARE) fused to a luciferase gene, and a plasmid encoding the androgen receptor (AR) cDNA sequence [31] (link). AR-EcoScreen™ cells were maintained in Dulbecco's modified eagle medium/F-12 media containing 10% fetal bovine serum. For the hypoxic condition, A549 cells were incubated at a CO₂ level of 5% with 1% O₂ balanced with N₂ using a hypoxic chamber (Thermo Fisher Scientific, Waltham, MA, USA).

Song H., Park J., Choi K., Lee J., Chen J., Park H.J., Yu B.I., Iida M., Rhyu M.R, & Lee Y. (2018). Ginsenoside Rf inhibits cyclooxygenase-2 induction via peroxisome proliferator–activated receptor gamma in A549 cells. Journal of Ginseng Research, 43(2), 319-325.

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Hypoxia and Reoxygenation in HBMECs

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HBMECs were washed three times with phosphate buffer solution (PBS) and incubated in Earle’s balanced salt solution (116 mmol/L NaCl, 0.9 mmol/L CaCl₂, 5.4 mmol/L KCl, 1 mmol/L NaH₂PO₄, 0.8 mmol/L MgSO₄, and 10 mg/L phenol red). In a hypoxic chamber (Thermo Scientific, USA), the compact gas controller maintained the oxygen concentration at 1% by injecting a mixture of 94% nitrogen and 5% carbon dioxide gas for 2 h. After treatment with hypoxia, the cells were transferred to complete culture medium with oxygen for 24 h. Normal control cells were incubated in a regular cell culture incubator under normoxic conditions.

Luo S., Li H., Mo Z., Lei J., Zhu L., Huang Y., Fu R., Li C., Huang Y., Liu K., Chen W, & Zhang L. (2019). Connectivity map identifies luteolin as a treatment option of ischemic stroke by inhibiting MMP9 and activation of the PI3K/Akt signaling pathway. Experimental & Molecular Medicine, 51(3), 37.

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Oxygen-Glucose Deprivation Induces Neuronal Injury

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The HT22 cells were obtained from Procell Life Science & Technology (China; CL-0595) and cultured in DMEM with 10% FBS at 37°C. To induce OGD, the neurons were cultured in glucose-free DMEM solution and maintained in a hypoxic chamber (Thermo Fisher Scientific) at 94% N₂, 1% O₂, 5% CO₂ and 37°C for 2, 4, 8, or 12 h, as previously described (Chen et al., 2011 (link); Xu et al., 2018 (link)). The HT22 cells in the normal group were subjected to OGD for 0 h. After OGD, the neurons were cultured in a normal DMEM solution with 10% FBS for 24 h or 48 h. After 24 h or 48 h of re-oxygenation, neurons were subjected to later assay.

Li J., Li B., Bu Y., Zhang H., Guo J., Hu J, & Zhang Y. (2022). Sertad1 Induces Neurological Injury after Ischemic Stroke via the CDK4/p-Rb Pathway. Molecules and Cells, 45(4), 216-230.

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Hypoxia induction in hASCs

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For induction of hypoxia, 80% confluent hASCs were plated in cell culture dishes. The serum-free media were degassed and then exposed in a hypoxic chamber (Thermo Fisher Scientific, Waltham, MA, USA) maintained below 5% CO₂, 5% H₂, and 1% O₂ at 37 °C. H9c2 cells were harvested after an 18 h incubation period and incubated with TRIzol Reagent (Life Technologies, Frederick, MD, USA) for quantification of miR expression.

Song B.W., Lee C.Y., Kim R., Kim W.J., Lee H.W., Lee M.Y., Kim J., Jeong J.Y, & Chang W. (2021). Multiplexed targeting of miRNA-210 in stem cell-derived extracellular vesicles promotes selective regeneration in ischemic hearts. Experimental & Molecular Medicine, 53(4), 695-708.

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