Antibiotic antimycotic

BMDMs were differentiated over 5–7 days in a medium with a recombinant macrophage colony-stimulating factor (M-CSF), as described previously [42 (link)]. The culture medium consisted of Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Waltham, MA, USA) and 1% antibiotic–antimycotic (Gibco™ antibiotic–antimycotic (100X); Gibco BRL, Waltham, MA, USA). Human retinal pigment epithelial ARPE-19 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM/F-12 (Welgene, Gyeongsan, Korea) with 10% FBS and 1% antibiotic–antimycotic.
The T. gondii RH strain was grown in ARPE-19 cells (MOI = 5) for 2–3 days at 37 °C and 5% CO₂. Host cells and parasites were washed with phosphate-buffered saline (PBS) after spontaneous host cell disruption. Protozoans were suspended in cold medium and passed through a 26-gauge needle and a 5.0 μm pore filter (Millipore, Billerica, MA, USA). The GFP-RH strain was kindly provided by Dr. Yoshifumi Nishikawa (Obihiro University of Agriculture and Veterinary Medicine, Japan). The T. gondii ME49 strain was obtained from the brain tissue of BALB/c mice that infected 50 cysts and were maintained every 3 weeks.

SH-SY5Y human neuroblastoma cells were grown in Roswell Park Memorial Institute 1640 medium supplemented with 10 % fetal bovine serum and 1 % antibiotic–antimycotic under an atmosphere of 5 % CO₂ and 95 % air (all from WELGENE). The medium was refreshed every three days. Cells below passage 24 were used for experiments. We chose the SH-SY5Y cell line because these human neuroblastoma cells express constantly endogenous tau.

HK-2 cells (Korean Cell Line Bank, Seoul, Korea) were cultured with RPMI 1640 (Welgene, Gyeongsan-si, Korea) supplemented with 10% FBS (Atlas Biologicals, Fort Collins, CO, USA) and 1% antibiotics-antimycotics (Genedirex, Taoyuan, Taiwan). The proliferation and survival of HK-2 was analyzed using a Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. THP-1 monocytes (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 (Welgene) supplemented with 10% FBS and 1% antibiotic–antimycotic solution. To differentiate THP-1 into M1 type, cells were stimulated with 200 ng/mL phorbol-12-myristate-13-acetate (PMA), 100 ng/ mL lipopolysaccharide (LPS), and 20 ng/mL IFNγ in RPMI 1640 medium supplemented with 10% FBS for 24 h. Subsequently, THP-1 monocytes were treated with 100 μg/mL of iMSC-EVs or pan PPAR-iMSC-EVs, 100 ng/mL LPS, and 20 ng/mL IFNγ in serum-free DMEM (Welgene, Gyeongsan-si, Korea). After 24 h, cells were subjected to RNA extraction and qPCR analysis.