Dulbecco s modified eagle s medium

BHK-21 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Procell, Wuhan China) with additional 10% FBS (Thermo Fisher, Waltham, MA, USA) at 37 °C with 5% CO₂, and the propagation of PRV was used. The parental virus of the PRV-AH strain had been identified as a mutant PRV [33 (link)]. The plasmids pMD-UA-EGFP-DA, which contained the homologous arms of the gE/gI gene and the EGFP expression cassette, and pMD-UA-gC-DA, which contained the homologous arms of the gE/gI gene and the gC-His expression cassette, were generated, respectively, in our previous study [34 (link)].

Tongue cancer cells (Cal27 and HN6) were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium (Procell, Wuhan, China) supplemented with 10 % fetal bovine serum (FBS) (Procell, Wuhan, China). The cells were incubated in a temperature-controlled incubator with 5 % CO₂.

The RSC96 rat SCs line was purchased from Procell Life Science & Technology (Wuhan, China), and they are primary cultured rat SCs that spontaneously transformed after extensive cultivation. Besides, mycoplasma identification by Hoechst staining, PCR, and routine culture techniques was negative, and the RSC96 cell line has been certified by ATCC and analyzed by STR. Cells were seeded in Petri dishes maintained in Dulbecco’s modified Eagle’s medium (Cat#PM150210, Procell) containing 10% fetal bovine serum (Excell Bio, Shanghai, China) and 1% penicillin/streptomycin in 5% CO₂ humidified atmosphere at 37 °C. The medium was replaced every 2 days. RSC96 cells were treated with α-syn PFF for 24 h, 36 h, and 48 h. To assess the role of TLR2, treated cells with the small molecule TLR1/2 inhibitor CU-CPT22 (8 µM) 1 h before α-syn PFF stimulation [38 (link)–40 (link)].

After euthanasia, the chest cavity was exposed, the lung tissue was washed with PBS containing 1% penicillin–streptomycin solution (Procell, Wuhan, China) mixture, and the lung tissue was cut into pieces and placed in 1 mg/mL collagenase type I (Gibco, Waltham, MA, USA), followed by a water bath at 37 °C for 1 h. After centrifugation, the lung tissue was resuspended and filtered through a 70 µm filter, and the filtered liquid was collected. The cells were lysed with red blood cell lysate (Solarbio) for 5 min, resuspended, filtered through a 40 µm filter, and the filtrate was collected. After centrifugation at 1500 rpm for 10 min, the cells were resuspended in high-glucose Dulbecco’s modified Eagle’s medium (Procell) containing 20% FBS (BI, Beit Haemek, Israel) and 1% penicillin–streptomycin solution. Passages 3–7 of primary lung fibroblasts were used in the experiment. Human lung fibroblasts (HFL-1) (Procell) were cultured in Ham’s F-12K medium (Procell) containing 10% FBS and 1% penicillin–streptomycin solution. All cells were cultured at 37 °C in a humidified incubator with 5% carbon dioxide.