2.4.2. Cell Culture and Experimental Conditions

Mouse macrophage-like cell line (J774A.1, Sigma) and BV2 cell line (murine microglia, kindly provided by Dr. Amy Ryan, SUNY Plattsburgh) were seeded at 3–5 <sup>×</sup> 105 cells/mL in 96 well plate (3–5 <sup>×</sup> 104 cells/well) using RPMI 1640 and DMEM medium (GIBCO), respectively. Both media were supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and l-glutamine. Both cell lines were cultured at 37 ◦C in a 5% CO2-supplemented atmosphere for at least overnight before the treatment with 10, 25, 50, and 100 μg/mL of CNCs. The peripheral blood mononuclear cells (PBMCs) were extracted from Leukotrap blood filters from healthy blood donors. The Leukotrap filters were obtained from UVM Health Network-CVPH North Country Regional Blood Center, Plattsburgh, NY. To retrieve blood cells, the filter was reverse flushed with 3 × 50 mL of calcium and magnesium-free PBS, followed by PBMCs isolation using a separation medium (LSM). Briefly, 4 mL of LSM was added to a 15 mL conical centrifuge tube and 6 mL of 1:1 diluted blood in calcium and magnesium-free PBS was carefully layered on LSM, creating a sharp blood-LSM interface. The conical tubes were centrifuged at 400× *g* at room temperature for 25 min. The top layer containing plasma was discarded and the mononuclear cell layer was carefully transferred to a clean tube and an equal volume of calcium and magnesium-free PBS was added. The cell suspension was centrifuged at 250× *g* to pellet cells and the cells were washed once in calcium and magnesium-free PBS and finally resuspended in RPMI medium for seeding. The PBMCs were seeded in 48 well plate at 1×10<sup>6</sup> cells/mL and cultured at 37 ◦C in a 5% CO2-supplemented atmosphere for at least overnight before the treatment with 10, 25, 50, and 100 μg/mL of CNC derivatives.
