Dmem f12 nutrient mixture

Human breast cancer cell lines MDA‐MB‐231, MCF7, MCF7/ADR, and benign breast epithelial cell line MCF‐10A were obtained from Chinese Type Culture Collection (Shanghai, China). MDA‐MB‐231 cells were cultured in Dulbecco's modified eagle medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37℃ humidified incubator with 5% CO₂. MCF7 and MCF7/ADR cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS at the same atmosphere mentioned above. MCF‐10A cell line was cultured in a 1:1 ratio of DMEM/F‐12 nutrient mixture (Gibco) medium supplemented with 10% FBS.

Jurkat cells (ATCC) and U937 cells (gift of Jeffrey D. Esko, UC San Diego) were maintained in RPMI 1640 media (Invitrogen) supplemented with 10% fetal bovine serum (FBS). HEK293t cells were obtained from ATCC and maintained in Dulbecco's modified Eagle medium (DMEM) with Glutamax (Invitrogen) supplemented with 10% FBS. Transient transfections of these cells with pcDNA3.1-S6-CCR7 and pcDNA3.1-CCR2-YFP were carried out using TransIT-LT1 reagent (Mirus Bio). HEK293s cells used for the scintillation proximity assay were stably transfected with an inducible pACMV-tetO-flag-CXCR4 plasmid and pcDNA6/TR (Invitrogen) according to the manufacturer's protocol and maintained in DMEM/10% FBS with blasticidin and G418. The Chinese hamster ovary cells (CHO-K1) and PGS745 cells (gift of Jeffrey D. Esko, UC San Diego) [23] (link) were maintained in DMEM/F12 nutrient mixture (Gibco) supplemented with 10% FBS. Transient transfections of CHO-K1 cells with pcDNA3.1-Flag-CXCR4, pcDNA3.1-CXCR4-GFP (gift of Adriano Marchese, Loyola University), pmEos2-CCR7 (with CCR7 placed C-terminally to mEos2), and CCR5-mCherry were carried out using the TransIT CHO transfection kit (Mirus Bio).

COS-7, HEK 293, HeLa, N2a and U-2 OS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) and RPE-1 cells were grown in DMEM/F-12 nutrient mixture (Gibco), all supplemented with 10% (v/v) fetal bovine serum at 37 °C in 5% (v/v) CO₂. Transient transfections were performed using 25 kDa linear polyethylenimine (Polysciences)^{36 (link)}. 18–48 h post-transfection the cells were subjected to microscopy.

ICR mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed at China Agricultural University. Mice with a vaginal plug in the next morning of mating were considered as 0.5 days post coitum (dpc), and the day after birth was considered as 1 dpp. Undamaged ovaries of 3 dpp mice were micro-dissected in cold phosphate-buffered saline (PBS) under a microscope. Isolated ovaries were cultured on 0.4 µm pore size inserts (Millipore, Cat#PICM0RG50) in 6-well culture plates (NEST, Cat#703002) for 4 days. Basal culture medium comprised 3 mL Dulbecco Modified Eagle Medium/Ham (DMEM)-F12 nutrient mixture (Gibco, Cat#C11330500BT) supplemented with penicillin-streptomycin (1:100, Gibco, Cat#15140-122). As a control, ovaries were treated with AIL (10nM, Selleck, Cat# S6885) or DMSO (Sigma, Cat#344282). Approximately half of the medium in each well was replaced with fresh medium every other day. Ovaries were maintained at 37°C under 5% CO2 and 95% air.

Primary microglial cultures were prepared from Sprague–Dawley rat pups on post-natal days 0–1. Microglia were acquired from primary mixed culture as described previously (Du et al., 2017 (link)). Briefly, the brain was removed from each newborn pup and the meninges stripped off in sterile ice-cold D-Hanks (in mM: 137.93 NaCl, 5.33 KCl, 0.44 KH₂PO₄, 0.34 Na₂HPO₄⋅12H₂O, 10.00 HEPES or NaHCO₃, and 5.56 D-glucose, pH 7.4). The tissue was dissociated by repeated aspiration, and the mixed cells were cultured with DMEM/F-12 nutrient mixture (Gibco) containing 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Microglial cells were separated after 14 days using the “shaking off” method. For Western blotting, microglia were plated at 2 × 10⁴ cells/well in 24-well plates. For reactive oxygen species (ROS) and lactate dehydrogenase (LDH) assays, microglia were plated at 5 × 10³ cells/well in 96-well plates. Microglia were allowed to adhere for 24 h before incubation with 1 μM sonicated WT or mutant PFFs for 2 or 24 h before experiments. For analysis of intracellular α-syn levels, cells were washed twice with 10 mM HCl and 150 mM NaCl for 2 min, followed by 2 ml PBS for 10 min, to remove fibrils adhering to the cell surface.