Heracell vios 250i co 2 incubator

Mouse Neuro-2A neuroblastoma (Neuro-2a) cells were established by R. J. Klebe and F. H. Ruddle with spontaneous tumours of strain A mice, and most of them were neuron-like with axon-like structures. Therefore, we purchased Neuro-2a cells (CL-0168) from Wuhan Procell for in vitro research. Neuro-2a cells were inoculated in MEM complete medium (PM170410B, Procell, China) mixed with foetal bovine serum. When the serum-free medium was needed in subsequent experiments, the medium was replaced with a pure MEM medium (PM170409B, Procell, China). The conventional cell culture was uniformly completed in a Heracell™ VIOS 250i CO 2 Incubator (Thermo Scientific™) at 37°C with 5% CO 2 .
An OGD cell model was constructed as described before [19] . Neuro-2a cells were adjusted to 1 × 10 5 cells/ml and then uniformly inoculated in a pure MEM medium. Neuro-2a cells in the control group were inoculated in MEM complete medium. The cells in the OGD group were transferred to a 37°C Heal Force 3-gas incubator (HF100) containing 1% O 2 , 5% CO 2 , and 94% N 2 . The subsequent OGD treatment was conducted with the continuous filling of an anoxic mixture for 6 hours. During this period, the cells in the control group were cultured routinely.

All fresh-collected specimens were obtained after informed consent of parents and patients and were anonymized for their analyses. This study was conducted in accordance to the local ethical committee approval (declaration number: DC-2017-3090). All patients and their paired-lines and xenografts are detailed in Table 1. As depicted in Figure 1 To prepare the 2D and 3D experiments, doubling time calculations were systematically done in an incubator (HERAcell VIOS 250i, CO2 incubator, Thermofischer), where oxygen concentrations were modulated from 1 to 21%. The cell growth was then followed for 7 days using IncuCyte ® Live Cell technology (Essen BioScience). Pictures were automatically acquired over time and quantified with IncuCyte ® software. Cell population doubling time was determined using the following equation: doubling time (days)=ln2(t-t0)/ln(Nt/N0), where t-t0 is the time of exponential growth, Nt and N0 are cell numbers at time t and t0, respectively.
For all experiments in single culture or in co-culture (see section 4.6), real-time scanned contrastphase images were acquired and analyzed by IncuCyte ® ZOOM Live Cell Analysis system (Essen BioScience).

Mouse Neuro-2A neuroblastoma (Neuro-2a) cells were established by R.J.Klebe and F.H.Ruddle with spontaneous tumors of strain A. white mice, and most of them were neuron-like with axon-like structures. Therefore, we purchased Neuro-2a cells (CL-0168) from Wuhan Procell Company for in vitro research. Neuro-2a cells were inoculated in complete MEM (PM150411B, Procell, China) mixed with fetal bovine serum. When serum-free medium was required in subsequent experiments, the medium was replaced with pure MEM (PM150410B, Procell, China). The conventional cell culture was uniformly completed in a Heracell™ VIOS 250i CO 2 Incubator (Thermo Scientific™) at 37°C with 5% CO 2 .
A OGD/R cell model was constructed by referring to Diao's method [ 18 ]. Neuro-2a cells were adjusted to a concentration of 1 × 10 5 cells/mL and then uniformly inoculated in pure MEM. Neuro-2a cells in the Control group were inoculated in complete MEM, and those in the OGD/R group were transferred to a 37°C Heal Force three-gas incubator (HF100) containing 1% O 2 , 5% CO 2 , and 94% N 2 . The subsequent OGD treatment was conducted with continuous filling of anoxic mixture for 6 hours (h). During this period, the cells in the Control group were cultured routinely. After 12 h of reperfusion, two groups of Neuro-2a cells were routinely cultured in complete MEM.