MLO-A5 cells were generously donated by the Bonewald Laboratory (University of Missouri-Kansas City) and were cultured in α-MEM containing nucleotides and L-glutamine (Life Technologies, Grand Island, NY) [38 (link)]. Media was supplemented with 5% fetal bovine calf serum (FCS) and 5% supplemented bovine calf serum (BCS) purchased from ThermoScientific Hyclone (Rockford, IL). Cells were plated into 100 mm2 culture plates (BD Biosciences, San Jose, CA) in a Revco Ultima II tissue culture incubator at 37°C and 5% CO2 (ThermoScientific, Ashenville, NC) and split every three days.
Revco ultima 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Revco Ultima II is a high-performance ultra-low temperature freezer designed for reliable long-term storage of samples in laboratory settings. It features a large capacity storage compartment with precise temperature control capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Hypoxia c chamber, by Biospherix (2 mentions)
Proox p110, by Biospherix (2 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Keratinocyte medium, by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium, by Lonza (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
4.5 g l glucose, by Thermo Fisher Scientific (1 mentions)
35 mm dishes, by Corning (1 mentions)
3 protocols using revco ultima 2
1
Cell Culture of MLO-A5 Osteocytes
2
Hypoxia and Normoxia Effect on Renal Cells
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hRCs were isolated and cultured using the established protocol in our laboratory (George et al., 2016 (link); Yim et al., 2019 (link)). The hRCs were cultured in GM containing a mixture of two types of media (1:1). One is DMEM/High glucose containing 10% FBS and 1% PS, and the other is keratinocyte medium (Thermo Fisher Scientific, Rochester, NY) supplemented with bovine pituitary extract, epidermal growth factor, 1% FBS, 0.08% insulin-transferrin-selenium (ITS; Lonza, Basel, Switzerland), and 1% PS. hRCs were seeded in 48-well plates (5,000 cells per well) and incubated in hypoxia (1% O2) or normoxia (21% O2) for 3 days with GM (positive control), SFM (negative control), CM (4×), single protein (ANGPTL4, follistatin, HGF, uPAR, or VEGF) or RPC (ANGPTL4 + follistatin + HGF + uPAR + VEGF) (Figure 1 ). For the normoxic culture, cells were cultured in a regular cell culture incubator with 21% O2 and 5% CO2. For the hypoxic culture, seeded cells were transferred in a hypoxia C-chamber (BioSpherix, Parish, NY) inside a standard CO2 incubator (Revco Ultima II; Thermo Fisher Scientific) with a compact gas oxygen controller (ProOx P110; BioSpherix) to maintain oxygen concentration at 1 and 5% of CO2 (Yim et al., 2019 (link)).
3
Hypoxia-Reoxygenation Rat Kidney Cell Assay
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The rat kidney tubular epithelial cell line (NRK-52E) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were maintained in DMEM containing 4.5 g/L glucose (Invitrogen) with 10% FBS at 37°C in a 5% CO 2 atmosphere and passaged twice a week. Cells were plated in 35-mm dishes (Corning Inc., Corning, NY, USA) at a density of 1 × 10 6 cells/dish, reached ~90% confluence by the next day, and were used for experiments. For hypoxia experiments, cells were treated in a hypoxia C-chamber (BioSpherix, Lacona, NY, USA) inside a standard CO 2 incubator (Revco Ultima II; Thermo Fisher Scientific) with a compact gas oxygen controller (ProOx P110; BioSpherix) to maintain oxygen concentration at 1% by introducing a gas mixture of 95% N 2 and 5% CO 2 for 24 h. After hypoxic treatment, the cells were washed with PBS (Gibco) and cultured with NM or iPSC-CM in 95% O 2 /5% CO 2 condition for 6 h (reoxygenation). Control cells were incubated in a regular cell culture incubator with 21% O 2 . At the end of the study, cells were monitored morphologically or harvested with indicated buffers to collect cell lysates for biochemical analyses.
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