All experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. Ten-day-old C57BL/6 mouse pups were collected from the Experimental Animal Center of Southern Medical University (Guangzhou, China) and NEC-like injury was induced as previously described [28 (link)] using formula [Similac Advance infant formula (Abbott Nutrition, Columbus, OH, USA):Esbilac (PetAg) milk replacer for Puppies, 2:1] containing enteric bacteria from a patient with surgical NEC (12.5 μl original stool slurry in 1 ml formula) via gavage five times daily. The mice were simultaneously exposed to hypoxic conditions (5% O2, 95% N2) for 10 min twice daily in a modular chamber (Billups-Rothenberg, San Diego, CA, USA) for 4 days. Pups were fed 50 μl/g body weight gavage over 2–3 min, using a single oral gavage via fine polyethylene tubing. For the inhibition of IL-6 upon the onset of NEC, mice were inoculated with 100 ng anti-IL-6 receptor (NEC + aIL6R group) or control IgG (NEC + cIgG group) antibodies via intraperitoneal injection once daily. According to our preliminary experiment (Fig. S2, a–c), control animals were left with their dams to breastfeed. Animals were euthanized on day 5 after NEC induction, or earlier if they demonstrated moribund signs.
Modular chamber
Manufactured by Embrient Inc
Sourced in United States
The Modular Chamber is a versatile piece of lab equipment designed for a variety of applications. It features a customizable and reconfigurable interior to accommodate different experimental setups. The chamber provides a controlled environment for conducting experiments, with options for temperature, humidity, and atmospheric gas composition regulation.
Automatically generated - may contain errors
Lab products found in correlation
Similac advance infant formula, by Abbott (1 mentions)
Glucose free dmem, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Huvecs, by Merck Group (1 mentions)
Egm 2 media, by Lonza (1 mentions)
Tgf β2, by Thermo Fisher Scientific (1 mentions)
Hcaec, by Merck Group (1 mentions)
Huvecs, by Lonza (1 mentions)
Hcaec, by Lonza (1 mentions)
7 protocols using modular chamber
1
Neonatal Necrotizing Enterocolitis Induction in Mice
2
Oxygen Glucose Deprivation in Hippocampal Neurons
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Oxygen glucose deprivation was performed as previously described with slight modification (19 (link)). Briefly, the primary hippocampal neurons were washed with glucose-free DMEM (Gibco, USA). Then, after the addition of glucose-free DMEM, they were placed in an anaerobic chamber containing 5% CO2 and 95% N2 at 37°C. We sealed cultures inside a modular chamber (Billups-Rothenberg) flushed for 10 min with the same premixed gas and placed inside an incubator for 3 h. OGD was terminated by replacing the exposure medium with neuronal culture medium added with Tat-HA-NR2B9c 250 nM, and returning the cells to a normoxic incubator to allow reperfusion for another 24 h. The cells cultured in the plain medium with ambient oxygen served as control (no exposure to OGD).
3
Ethanol Effects on Alveolar Macrophages
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The mouse alveolar macrophage cell line, MH-S (ATCC; Manassas, Virginia, United States), was utilized to study the effects of ethanol in vitro. MH-S cells were cultured for 24 h in RPMI-1640 medium containing 10% FBS plus 1% penicillin/streptomycin, followed by treatment ±0.08% (17 mM) ethanol for 3 days ± 10-μM pioglitazone (PIO) during the last 24 h of ethanol exposure (Wagner et al., 2012 (link)). Ethanol-stimulated cells were incubated in a modular chamber (Billups-Rothenberg, Inc.; Del Mar, California, United States) to prevent alcohol evaporation. MH-S cells were collected for analysis 24 h following the last control or ethanol treatment. Exposure of MH-S cells to 0.08% or 17-mM ethanol for 3 days results in similar experimental findings as AM from our in vivo mouse model of chronic ethanol ingestion and AM isolated from human subjects with a history of AUD (Morris, Harris, Brown, & Yeligar, 2021 (link); S. M. Yeligar et al., 2012 (link), 2014 (link); S. M. Yeligar, Mehta, et al., 2016 (link)).
4
Hypoxia Exposure and Cell Incubation
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Cells were maintained in D-Hanks solution and seeded into a sealed chamber (Modular Chamber, Billups-Rothenberg, Del Mar, CA) that was filled with 95% N2/5% CO2. Ventilation was performed at a rate of 5 l/min for 15 min, until the concentration of oxygen in the chamber was lower than 1% [13 (link), 14 (link)]. After incubation at 37°C for 2 h, the D-Hanks solution was removed, and high glucose DMEM containing 10% FBS was added, followed by incubation with 5% CO2 at 37°C for 3 h.
5
Endothelial-to-Mesenchymal Transition Induction
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HUVECs (Lonza, Basel, Switzerland) and HCAECs (Lonza) were cultured in EGM-2 media (Lonza). To induce EndMT, six-well plates were pre-coated with 31.25 ng ml−1 fibronectin (F2006; Sigma) for 1 h. HUVECs or HCAECs were initially seeded at 5,000 or 7,500 cells cm−2, respectively. Growth medium was changed for complete growth medium containing 50 ng ml−1 TGF-β2 (#100-35; Peprotech, Rocky Hill, NJ) with or without hydrogen peroxide (H2O2) (H1009; Sigma) at 100 or 200 μM (as indicated)58 (link) the following morning and every other day for 5 days.
Endothelial cell culture under hypoxic conditions was performed by incubating the cells in an airtight Plexiglass chamber (Billups-Rothenberg Modular Chamber; Del Mar, CA) with an atmosphere of 5% CO2/95% N2 at 37 °C, as described59 (link). Rather than changing growth media every other day, to minimize cell exposure to oxygen the media was replaced only once during the 5-day experiment. Media was also changed only once during the 5-day experiment for control (comparator) cells grown under non-hypoxic conditions.
Endothelial cell culture under hypoxic conditions was performed by incubating the cells in an airtight Plexiglass chamber (Billups-Rothenberg Modular Chamber; Del Mar, CA) with an atmosphere of 5% CO2/95% N2 at 37 °C, as described59 (link). Rather than changing growth media every other day, to minimize cell exposure to oxygen the media was replaced only once during the 5-day experiment. Media was also changed only once during the 5-day experiment for control (comparator) cells grown under non-hypoxic conditions.
6
Ethanol Effects on Alveolar Macrophages
7
Ethanol effects on alveolar macrophages
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