U87-MG cells were cultured in DMEM with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at 37°C in a humidified 5% CO2, 95% air incubator. PC12 cells were cultured in DMEM with 5% FBS, 10% horse serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at 37°C in a humidified 10% CO2, 90% air incubator. Before hypoxic exposure, the media was replaced with fresh media containing 25 mM HEPES, then the cells were placed in a modulator incubator chamber (Billups-Rothenberg) that was flushed with a gas mixture consisting of 1% O2, 5% CO2, with balance N2, and incubated at 37°C. The HIF inhibitors digoxin [16 (link)] and acriflavine [17 (link)] were purchased from Sigma; cells were pre-treated with inhibitors for 1 h before hypoxic exposure. Control cells were treated with vehicle (0.1% [v/v] DMSO).
Modulator incubator chamber
Manufactured by Embrient Inc
Sourced in United States
The Modulator incubator chamber is a laboratory equipment device designed to maintain a controlled environment for various experimental purposes. It provides precise temperature and humidity regulation to support the growth and development of cell cultures, microorganisms, or other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Digoxin, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Acriflavine, by Merck Group (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Cocl2, by Merck Group (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
24 well cell culture plate, by BD (1 mentions)
7 protocols using modulator incubator chamber
1
Hypoxia Induction and HIF Inhibition
2
Hypoxic Cell Culture Protocol
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Cells were cultured to -80% confluency, and then transferred to hypoxic conditions, using a modulator incubator chamber (Billups-Rothenberg, Del Mar, CA, USA), which was flushed for 10 minutes with a gas mixture containing 95% N2/5% CO2, resulting in a final concentration of 3% O2. The incubator chambers were sealed and placed in a traditional incubator at 37℃.
3
Generating Bone Marrow Derived Macrophages
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Bone marrow derived macrophages (BMDMs) were generated from 6-8 week old C57BL/6 mice as described before (43 (link)). Briefly, the bone marrow derived cells were collected by flushing the femurs and tibias of the mice with PBS using a 30-gauge needle. Following treatment with red cell lysis buffer, purified cells were cultured in RPMI + 10% FBS + 50 ng/ml MCSF (Peprotech) for 5-7 days before use. For hypoxia experiments, BMDMs were placed in a modulator incubator chamber (Billups-Rothenberg) under 1% O2 conditions as previously described (41 (link), 43 (link)).
4
Hypoxia Induction in Melanoma Cells
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For the induction of hypoxia, melanoma cell cultures were incubated for 24 (B16F10) to 72 h (Mel3) in a Modulator Incubator Chamber (Billups-Rothenberg, Inc., San Diego, CA, USA) in a humidified atmosphere containing 5% CO2, 1% O2, and 94% N2 (special gas cylinder supplied by Linde Chile) at 37 °C. In some experiments, CoCl2 (Sigma-Aldrich, St. Louis, MO, USA), a chemical inductor of hypoxia, was used at 150 μM final concentration. Hypoxia induction was routinely tested by flow cytometry using Hypoxia Green Reagent (HGR, Invitrogen, Paisley, UK) according to the manufacturer instructions. HGR is a membrane-permeant probe that releases rhodamine as O2 levels decrease, resulting in a fluorogenic response.
5
Cell Line Characterization and Manipulation
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All cell lines were obtained from the American Type Culture Collection and authenticated in 2018 using short tandem repeat analysis. Hs578T and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) in a humidified incubator equilibrated with 5% CO 2 at 37°C, whereas MDA-MB-231 and MDA-MB-468 cells were maintained in L-15 medium without CO 2 . All media were supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin. For all hypoxic conditions, cells were placed in a modulator incubator chamber (Billups-Rothenberg, San Diego, California, USA) flushed with 1% O 2 . Transfections were carried out using Lipofectamine® RNAiMAX Reagent (Invitrogen) following the manufacturer's instructions. Each experiment was performed in triplicate. For RNAi experiments, at least three independent shRNA sequences were tested for each gene, and the two with the highest efficiency were used. The shRNA sequences used in this study are listed in Table S1.
6
Murine Macrophage Isolation and Hypoxia Experiments
7
Rearing Honeybee Larvae under Controlled Conditions
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Apis mellifera larvae were collected from nine hives near Fargo, Cass County, North Dakota during a three-week period in the summer of 2015. Hives were supplemented with pollen patties (Mann Lake, MN, USA) and a 1 : 1 sucrose–water solution (Brushy Mountain Bee Farm, NC, USA) during poor foraging conditions. First instar larvae (0–21 h old) were transferred into 24-well cell culture plates (Falcon, Corning, Durham, NC) and placed onto 10 µl of diet. The 24-well plates were stored inside a modulator incubator chamber (Billups-Rothenberg, del Mar, CA, USA). Larvae were kept at a constant 34°C, darkness and relative humidity (RH) of 96% using potassium sulfate (K2SO4) [33 (link)]. Larvae were fed according to treatment in a factorial design of nine diet qualities and eight quantities with an additional ad libitum treatment for the medium diet, as described in the following sections. At the prepupal stage they were moved into 24-well cell culture plates containing Kimwipes (Kimtech Science, USA) sterilized in EtOH [34 ]. Pupae were maintained at a constant 34°C, darkness and 75% RH using NaCl until adult eclosion. Adults were stored at −20°C.
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