For inducing H-MSCs, MSCs derived from the 4th passage were incubated under 5% O2 condition in a hypoxia incubation chamber (STEMCELL Technologies, Biopolis, Singapore) for 24 h at 37°C and 5% CO2, then collected for the following experiment. The IPAs animal model was treated by 3 x 106 as a high dose (T1) and 1,5 x 106 as a low dose of H-MSCs (T2) via submucosal injection, whereas the Sham group received NaCl only and Control group received the omental patch treatment.
Hypoxia incubation chamber
Manufactured by STEMCELL
Sourced in Singapore, Canada
The Hypoxia Incubation Chamber is a laboratory equipment designed to provide a controlled oxygen environment for cell culture experiments. It maintains a specified oxygen level within the internal chamber, allowing researchers to simulate hypoxic or anoxic conditions as required for their study.
Automatically generated - may contain errors
3 protocols using hypoxia incubation chamber
1
Hypoxia-Induced Mesenchymal Stem Cells in IPA
2
Neuroprotective Effects of PAP-1 and Minocycline in Hippocampal Slices
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Hippocampal slice cultures (400 μm thick) were prepared from 7‐day‐old C57BL/6J mice as described.28 After 3 days, culture medium was changed to a hypoxic/hypoglycemic medium (75% Neurobasal, 25% HBSS, 1% L‐glutamine, bubbled with 99.4% nitrogen). Slices were placed in a hypoxia incubation chamber (Stemcell Technologies, Vancouver), and nitrogen gas was flowed into the chamber, followed by incubation at 37o for 1 h. The culture medium was replaced by normal culture medium containing glucose, and slices were placed in a tissue culture incubator. After 2 h of culture, 1 μmol/L of PAP‐1 or 10 μmol/L of minocycline were added and slices cultured for 3 days. Slices were fixed in 4% formaldehyde and stained with anti‐NeuN (1:400; Chemicon) and anti‐Iba1 (1:400; Wako Chemical) followed by secondary Alexa‐Fluor®488‐conjugated anti‐mouse or Alexa‐Fluor®568‐conjugated anti‐rabbit antibodies (1:700; Life Technologies). For Western blotting, slices were lysed and protein electrophoresed and visualized as previously described.14 The following primary antibodies were used as follows: anti‐PSD95 (1:1000) and anti‐GAPDH (1: 3000; both Cell Signaling Technology). The secondary antibody was an HRP‐conjugated anti‐rabbit antibody (1:3000; Amersham Biosciences).
3
Clonogenic Assay under Hypoxic Conditions
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Cells were counted, resuspended at 2 × 103 cells/mL in the medium (DMEM with 10% FBS and L-glutamine) containing 0.3% weight/volume (w/v) agar (Bacto, Dickinson, Sparks, MD, USA) and overlaid onto a 30-mm dish containing a solidified bottom layer of 0.6% w/v agar in the same medium. After incubation for 10–15 days at 37 °C and 10% CO2, all dishes were stained by adding 1 mL/dish of 0.01% (w/v) crystal violet (Fronine, Taren Point, NSW, Australia), and the colonies were counted with a dissection microscope. The assaying was triplicate. The role of cultivation under hypoxic conditions was analyzed in hypoxia incubation chamber (StemcellTechnologies, Vancouver, British Columbia, Canada) with certified medical grade pre-mixed gas (1% O2, 5% CO2, 94% N2).
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