Breast cancer cell lines MCF-7 (NCI-DTP Cat# MCF7, RRID:CVCL_0031) cells were maintained in RPMI-1640 supplemented with 10% FBS, 1% Sodium pyruvate, 1% Pen-Strep. MDA-MB-231 (NCI-DTP Cat# MDA-MB-231, RRID:CVCL_0062) cells were cultured and maintained in DMEM supplemented with 10% FBS, 1% Sodium pyruvate, 1% Pen-Strep in a humidified 5% CO2 incubator (ESCO Cell Culture incubator, USA). For all hypoxic exposure, cells were maintained under 1% oxygen using Whitley H35 Hypoxystation (Don Whitley Scientific Limited). For chronic hypoxia, the cells were maintained under hypoxia for 9 continuous days and for intermittent hypoxia, the cells were cyclically maintained in 1% oxygen for 24 h followed by maintenance in 21% oxygen for 24 h. After the fifth cycle of hypoxia, cells were collected for all experiments.
Whitley h35 hypoxystation
Manufactured by Don Whitley Scientific
Sourced in United Kingdom
The Whitley H35 Hypoxystation is a controlled environment workstation that provides a stable, low-oxygen atmosphere for cell culture and research applications. It maintains precise oxygen, carbon dioxide, and temperature levels within the enclosed workspace.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Str profiling, by Eurofins (2 mentions)
Powerplex 21 system, by Promega (2 mentions)
Mg132, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
H1299, by National Collection of Authenticated Cell Cultures (1 mentions)
Originpro version 8, by OriginLab (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Bv2 cells, by American Type Culture Collection (1 mentions)
Fibroblast medium, by ScienCell (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Heracell, by Thermo Fisher Scientific (1 mentions)
Tapi 2, by Merck Group (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
Penstrep solution, by Corning (1 mentions)
28 protocols using whitley h35 hypoxystation
1
Breast Cancer Cell Line Hypoxia Models
2
Hypoxic Cultivation of NSCLC Cell Lines
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The human NSCLC cell lines H1299 (p53-null), Calu-1(p53-null) and H460 (wild-type p53) were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The H1299 and Calu-1 cell lines were cultured in RPMI 1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) and H460 was cultured in DMEM medium (HyClone), both being supplemented with 10% fetal bovine serum (FBS, HyClone), 100 μg/mL streptomycin (Gibco, Carlsbad, CA, USA) and 100 U/mL penicillin (Gibco). For normoxic cultures, the cells were maintained at 37 °C in a humidified incubator with 5% CO2 and 95% air. For hypoxic cultures, cells were maintained at 37 °C in a Whitley H35 Hypoxystation (Don Whitley Scientific, Shipley, UK) with 1% O2, 94% N2 and 5% CO2. All cell lines were free of mycoplasma. MCL (Wuhan ChemFaces Biochemical, Wuhan, China), protein synthesis inhibitor Cycloheximide (CHX, Sigma, St Louis, MO, USA), and proteasome inhibitor MG132 (Sigma) were dissolved in DMSO.
3
Hypoxia Sensitizes Cells to Olaparib
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Exponentially growing cells were seeded into 6-well plates, and exposed to 21% O2 (normoxia) or 1% O2 (hypoxia) in a Whitley H35 Hypoxystation (Don Whitley Scientific Ltd) for 16 hours. An amount of 5 μmol/L olaparib (Selleck) or 0.05% DMSO was added to the medium 1 hour before irradiation (0-6 Gy) with a cesium-137 source at 1.69 Gy/min (GSR D1 irradiator; Gamma Service Ltd) under normoxic or hypoxic conditions. Twenty-four hours after irradiation, cells were washed, and fresh medium was added. Plates were incubated for 6 to 13 days to allow colonies to form. Colonies were then fixed and stained with 0.5% crystal violet in 5% acetic acid and 75% methanol. Colonies (≥50 cells) were counted. Clonogenic survival curves and the sensitization enhancement ratio at 50% (SER50) were generated using OriginPro version 8.5.1 software (OriginLab Corp).
4
Rat Cardiomyocyte Hypoxia-Reoxygenation Model
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Rat cardiomyocyte-derived cell line H9c2 was purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% v/v fetal bovine serum (FBS) and 1% v/v penicillin/streptomycin at humidified atmosphere containing 5% CO2 at 37°C. Hypoxic treatment was conducted by incubating cells in glucose-free DMEM under conditions of 94.9% N2/5% CO2/0.1% O2 for 12 h in a hypoxystation (Whitley H35 hypoxystation, Don Whitley Scientific), and then reoxygenation was carried out by incubating cells in growth medium for 6 h under normoxic conditions, as described above. All of the drugs were added into the medium before and retained in the culture medium over the entire period of reoxygenation.
5
Cell Culture Conditions and Validation
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HeLa, HEK293T, MCF7, A549 cells and skin fibroblasts cells (gift from the Larrieu laboratory, Cambridge Institute for Medical Research) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) and supplemented with 10% fetal calf serum (FCS). 786-0 cells and 786-0 VHL reconstituted cells48 (link) were maintained in RPMI-1640 (Sigma) supplemented with 10% FCS. Hypoxic cell culture was performed in a Whitley H35 Hypoxystation (Don Whitley Scientific) at 37°C, 5% CO2, 1% O2 and 94% N2. Cells were confirmed mycoplasma negative (Lonza, MycoAltert), and authenticated by STR profiling (Eurofins Genomics). Full details of reagents and antibodies used as shown in Supplementary Table 2 .
6
Cell Culture Conditions and Validation
7
Mimicking Ischemic Conditions in vitro
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To mimic ischemic-like conditions in vitro, we subjected rPMVECs to OGD/R as described (Shen et al., 2004 (link)). Briefly, rPMVECs were washed three times with PBS, fed serum- and glucose-free medium (Gibco), and then placed for 1 h at 37°C in a Whitley H35 Hypoxystation (Don Whitley Scientific) in an atmosphere of 1% O2, 5% CO2, and 94% N2. Then, cells were cultured for 4 h at 37°C in glucose-containing medium in an atmosphere of 5% CO2 and 95% O2.
8
Hypoxia-Induced Macropinosome Formation
9
Retinal Neurons and Microglial Responses
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R28 cells, an E1A immortalized model of retinal neurons, were a generous gift from Gail M. Seigel (State University of New York). The R28 cells were cultured in DMEM with low glucose (5 mM), 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C under 5% CO2 in a humidified incubator. R28 cells were plated for 48 h and then divided into normal control group (N) and glyoxal‐treated group (G).
BV2 cells, a mouse microglial cell line, were purchased from American Type Culture Collection and cultured in DMEM with high glucose (25 mM), 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C under 5% CO2 in a humidified incubator. After 24 h culture, the cells were divided into three groups: normal control (N), hypoxia treatment (H) and hypoxia+FKN treatment (H+F). The latter two groups were incubated in a hypoxic workstation (Whitley H35 Hypoxystation; Don Whitley Scientific, Bingley, UK) with 1% O2 with or without FKN treatment.
BV2 cells, a mouse microglial cell line, were purchased from American Type Culture Collection and cultured in DMEM with high glucose (25 mM), 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C under 5% CO2 in a humidified incubator. After 24 h culture, the cells were divided into three groups: normal control (N), hypoxia treatment (H) and hypoxia+FKN treatment (H+F). The latter two groups were incubated in a hypoxic workstation (Whitley H35 Hypoxystation; Don Whitley Scientific, Bingley, UK) with 1% O2 with or without FKN treatment.
10
Hypoxic Fibroblast Response to Pirfenidone
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HPAAFs were purchased from ScienCell (CA, USA) and grown in fibroblast medium (ScienCell, CA, USA) consisting of 2% fetal bovine serum (FBS), 1% fibroblast growth supplement, and 1% penicillin/streptomycin. Cells were grown at 37°C in a 5% CO2 incubator and used between passages 3 and 8. The cells were exposed to hypoxic conditions inside a standard culture chamber (Whitley H35 Hypoxystation, Don Whitley Scientific, England), with an atmosphere of 5% O2, 5% CO2, and 90% N2. Pirfenidone (PFD) was added to cells at a concentration of 0.1mg/ml or 0.2mg/ml for 12 h or 24 h before hypoxia.
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