Hmc3 cells

HMC3 cells (ATCC; Manassas, VA, USA) were cultured in EMEM containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin with 5% CO₂ at 37 °C. HRMECs (Cell Systems; Seattle, America) were cultured in Complete Classic Medium with Serum & CultureBoost (Cell Systems) at 37 °C in 5% CO₂. All cell lines were authenticated and confirmed to be mycoplasma free using a PCR Mycoplasma Test Kit (Applied Biosystems, 4460623). For coculture experiments, HMC3s were precultured under the corresponding conditions for 24 h (DCA was added at a concentration of 20 mM; rotenone was added at a concentration of 50 nM; A-485 was added at concentrations of 5, 10, and 20 μM according to previous studies) and then cocultured with HRMECs.

K562 cells were a gift from Dr. Yun (Nancy) Huang lab from Texas A&M University Health Science Center Houston, and were originally purchased from ATCC. They were cultured in RPMI 1640 medium (CORNING) supplemented with 10% fetal bovine serum. HeLa, MCF7 and 293T cells were purchased from ATCC, and cultured in DMEM medium (CORNING) supplemented with 10% fetal bovine serum. HMC3 cells are immortalized primary human embryonic microglia cells, and were purchased from ATCC. They were cultured in EMEM medium supplemented with 10% FBS. hNPCs were generated by an in-house protocol (see below). Mycoplasma was examined every 6 months to a year.

HMC3 cells were purchased from ATCC (Manasass, VA, United States). The cells were cultured in minimum essential medium (MEM) supplemented with 10% FBS (Hyclone, Logan, UT, US), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Waltham, MA, United States) and maintained in a humidified atmosphere of 5% CO₂ in air at 37°C. The cells were trypsinized in 0.25% trypsin-EDTA (Gibco) and maintained at 2 × 10⁴ cells/cm² once they reached maximum confluency.

CW468 cells were established by Dr. Jeremy N. Rich lab (UCSD) and cultured in Neurobasal medium (NBM, Gibco, Cat# 21103049) supplemented with 1% B27 minus vitamin A, 1% _L-glutamine, 1% sodium pyruvate, 1% penicillin/streptomycin (P/S), 10 ng/mL basic human fibroblast growth factor (Peprotech, Cat# AF-100-18B), and 10 ng/mL human epidermal growth factor (Peprotech, Cat# AF-100-15). THP1 cells (American Type Culture Collection, ATCC, Cat# TIB-202) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 1% P/S. HMC3 cells (ATCC, Cat# CRL-3304) were cultured in Minimum Essential Medium (MEM, Gibco) medium with 10% fetal bovine serum (FBS), 1% P/S, and 1% non-essential amino acids. HUVEC (Cell Applications, Cat# 200p-05n) were cultured in endothelial cell growth medium v1 (Cell Applications, Cat# 211-500) supplemented with 1% P/S. U251 cells were cultured in DMEM medium with 10% FBS and 1% P/S. All cells were maintained at 37 °C with 5% CO₂ and passaged with TrypLE (Gibco) or Accutase (Stemcell Technologies) every two to three days. Cell line authentication was conducted for immortalized cell lines, including HMC3 and THP1, using short tandem repeat (STR) profiling and comparison to known reference profiles to ensure data integrity and reliability.

HMC3 cells were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA, CRL-3304). The HOG cell line was a kind gift of Dr. José Antonio López Guerrero (University of Madrid, Spain). The HMC3 and HOG cells were grown in EMEM (Lonza, BE12-662F) with 10% heat-treated FBS (Gibco, 10270106), 1% glutamax and 1% Pen-Strep, and passaged twice per week at a 1:20 splitting ratio (maximum passage number 35). The medium was refreshed every other day. Mycoplasma testing of the cell lines was performed tri-annually.