For hypoxic treatments, we applied the BIONIX-1 hypoxic culture kit (Sugiyamagen, Tokyo, Japan) as previously described [42 (link), 43 (link)]. This system consists of an AnaeroPack-Anaero 5% (oxygen absorber; Mitsubishi Gas Chemical, Tokyo, Japan), an OXY-1 oxygen monitor (JIKCO, Tokyo, Japan), an AnaeroPouch (Mitsubishi Gas Chemical), and plastic clips for sealing the pouch. Briefly, PCAs, the oxygen absorber, and the oxygen monitor were arranged in the pouch, and the left open side was sealed with a clip. The O2 concentration in the pouch rapidly decreased after sealing; once a low O2 concentration (approximately 0.1%) was reached, the pouch was sealed with another clip between the culture dish and the oxygen absorber to stop further oxygen absorption. Then, the pouch was maintained in an incubator at 37°C.
Anaeropack anaero 5
Manufactured by Mitsubishi
Sourced in Japan
The AnaeroPack-Anaero 5% is a laboratory equipment product designed to create an anaerobic environment with 5% carbon dioxide concentration. Its core function is to provide a controlled atmospheric condition for the cultivation and growth of anaerobic microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Anaeropouch, by Mitsubishi (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
Deferoxamine mesylate, by Abcam (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (1 mentions)
Anaeropack system, by Mitsubishi (1 mentions)
4 protocols using anaeropack anaero 5
1
Hypoxic Cell Culture Technique
2
Hypoxia and Radiosensitivity of Spheroids
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To investigate the effect of oxygen tension on radiosensitivity, spheroids formed as described above were treated using the AnaeroPack‐Anaero 5% (Mitsubishi Gas Chemical, Tokyo, Japan) as described previously.17 Incubation time was 9 h, which was sufficient to generate severe hypoxia (pO2 < 0.1% = 0.78 mmHg) in the liquid phase after equilibration with the air phase. Irradiation was given under this condition and then the spheroids were returned to normoxia. Twenty‐four hours after reoxygenation, the spheroids were disaggregated and red and green cells were sorted as described above. Radiosensitivity was determined by a colony‐forming assay as described below.
3
EHEC Anaerobic and Aerobic Culture
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For anaerobic culture, EHEC strains grown overnight [the optical density at 600 nm (OD600) = 1.0 ~ 1.5 × 109 cfu/ml] were diluted 1:100 with LB broth containing 10 mmol/L HEPES (pH 7.0) (LB‐pH 7.0) and grown statically at 37°C using Anaero Pack‐Anaero 5% (Mitsubishi Gas Chemical Company, Tokyo, Japan). For aerobic culture, EHEC strains grown overnight (OD600 = 1.0 ~ 1.5 × 109 cfu/ml) were diluted 1:100 with LB‐pH 7.0 and grown statically at 37°C under aerobic conditions.
4
Hypoxia-induced Lung Cancer Cell Culture
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Human lung adenocarcinoma cell line A549 and pulmonary squamous cell carcinoma cell line LK-2 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan), respectively. All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Kyoto, Japan), supplemented with 10% fetal bovine serum (FBS) as well as 100 U/ml penicillin G, 0.1 mg/ml streptomycin and 0.2 mg/ml amphotericin B (all from Gibco-BRL; Thermo Fisher Scientific, Inc., Tokyo, Japan), at 37°C in a humidified atmosphere of 5% CO2. For cell culture under hypoxic conditions, the AnaeroPack system (AnaeroPack-Anaero 5%; Mitsubishi Gas Chemical, Tokyo, Japan) was used in conjunction with an OXY-M oxygen monitor (JIKCO, Tokyo, Japan). To mimic a hypoxic condition, 200 µM of deferoxamine mesylate (BioVision, Inc., San Francisco, CA, USA) was added to the culture medium.
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