Proox model p110

The HL-1 cells were cultured in Claycomb medium com-pleted with 10% fetal bovine serum (Euroclone, Milan, Italy), 2 mM L-glutamine, 0.1 mM norepirephrine, and 100 μg/mL penicillin/streptomycin (Lonza, Basel, Switzerland) under 0.2% hypoxia for 24 h using ProOx Model P110 (BioSpherix, 25 Union Street, Parish, NY 13131) hypoxia chamber as per the referred manual of ProOx Model P110. In CTRL sample, after 24 h of acute hypoxia stimulus, the cells were successively cultured in normoxia for the next 24 h at 37°C in a humidified atmosphere of 5% of CO₂ in air. In the sample entitled HL-1 + EVs-hGMSCs UN, the HL-1 cells were maintained under acute hypoxic stimulus at 0.2% for 24 h and then treated with EVs derived from hGMSCs under normoxia for 24 h. In the sample entitled HL-1 + EVs-hGMSCs UH, the HL-1 were first treated with EVs derived from hGMSCs and kept in hypoxia at 0.2% for 24 h and then in normoxia for 24 h.

A hypoxia chamber (ProOx Model P110, BioSpherix, Parish, NY, USA) was used to create a hypoxic laboratory environment in which oxygen was reduced by nitrogen gas (HP, Airgas, Hyattsville, MD, USA). At 30 hpf larvae were exposed to hypoxia (1 mg/L oxygen) for 18 hr or to normoxia (outside of the chamber). Larvae were placed individually in wells of a 24-well plate (CytoOne, USA scientific, Ocala, FL, USA) containing 2 ml clean fish system-water. Twenty-four larvae were used per treatment for surface fish and 48 larvae for cavefish from two separate clutches of eggs per morph. To measure post-anal tail growth, each larva was imaged before placement in a well and again at the end of the treatment using a microscope as described above. Image-J was used to measure post-anal tail length (Figure 6A) and growth determined as the differential between the two time points. To measure eye size, eye diameter was measured after the 18 hr. hypoxia or normoxia treatment with ImageJ. After imaging, larvae were stained with o-dianisidine and blood cells were counted from images as described above.

The cultured cells of both MSC-lung and MSCs-CPAM were seeded in a 6-multiwell plate with a density of 8 × 10⁴ for each well. After seeding cells were maintained for 24 h in the standard medium MSCBM (Lonza) in incubator at 37°C with a humidified atmosphere at 5% CO₂. Then cultured cells of MSC-lung and MSCs-CPAM were subjected to hypoxic treatment by inserting the plates into the ProOx Model P110 (BioSpherix, New York, NY, United States) hypoxia chamber for 24 h at 0.2% of hypoxia. Cells were observed under inverted light microscopy (Leica Microsystem, Milan, Italy) to evaluate the morphological features in normoxic and hypoxic conditions. Cultured cells of MSC-lung and MSCs-CPAM were maintained in incubator with normoxic condition and used as control cells.