12 well culture plates

In mimicking the hypoxia condition, a chamber (Model C374, BioSpherix, Union St Parish, NY USA) with adjustable mixture of oxygen and carbon dioxide gasses (Linde Malaysia Sdn. Bhd., Malaysia) was used. The astrocyte cells were plated on 12-well culture plates (Cat no: 0030721012, Eppendorf AG, Harmburg, Germany) with a 15 mm cover slip (EMS, Pennsylvania USA) and then were exposed to hypoxic condition via hypoxia chamber. At first, the cells were exposed to hypoxia at various time points; 5 min, 10 min, 15 min, 20 min, 25 min and 30 min at hypoxia, 3% O₂. Then, 15 min was chosen as the time point. The cells were then exposed to different oxygen concentrations; 15% O₂, 10% O₂, 5% O₂ and 3% O₂ in 15 min (16 (link), 17 (link)).

The spleen was isolated from each minipig exposed to FA. Single splenic cells were prepared as described previously^{46 (link)}. For primary cell culture, the single splenic cells were suspended in RPMI 1640 medium (Lonza, Walkersville, MD, USA) containing 10% heat-inactivated foetal bovine serum (Gibco, Grand Island, NY, USA), penicillin (100 U/mL) (Gibco), and streptomycin (100 μg/mL) (Gibco). The splenocytes were seeded at a density of 1 × 10⁶ cells/100 µL/well in 12-well culture plates (Eppendorf, Hamburg, AG, Germany) and incubated for 72 h with 2.5 μg/mL Con A (Sigma-Aldrich Co.) at 37 °C with 5% CO₂.
For flow cytometric analysis, the splenic single cells were resuspended in Flow Cytometry Staining Buffer (eBioscience Inc., San Diego, CA, USA) prior to analysis.

Cells were seeded into 12-well culture plates (Eppendorf) at the density of 2 × 10⁴/cm², cultivated for 24 h before the addition of dendrons and bola dendrimers. After 96 h, the cells were harvested and counted in the Z2 particle counter (Beckman-Coulter, Indianapolis, IN, USA) to calculate proliferation kinetics. For the estimation of pro-apoptotic potential of the compounds, the cells were seeded at the density of 2 × 10⁵/cm², and treated as described above for 48 h. Subsequently, AnnexinV/Propidium iodide assay was performed (FITC AnnexinV Apoptosis Detection Kit, BD Pharminogen™, San Diego, CA, USA) using ImageStreamX^® cytometer (Amnis Corp, Seattle, WA, USA) as described by Ryszawy et al. [42 (link)]. Data were analyzed with dedicated IDEAS^® 6.2 software (Amnis Corp, Seattle, WA, USA).