High glucose dulbecco s modified eagle s medium

Murine melanoma cell line B16 and OVA-B16, murine hepatocarcinoma cell line H22, and murine macrophage cell line Raw264.7 were purchased from China Infrastructure of Cell Line Resources (Beijing, China) and cultured with RMPI-1640 (Life Technologies) or high glucose-Dulbecco’s modified Eagle’s medium (Corning) medium supplemented with 10% feral bovine serum (FBS) (Gibco, Australia) at 37 °C with 5% CO₂. Cells were tested for mycoplasma detection, interspecies cross-contamination, and authenticated by isoenzyme and short tandem repeat analyses in Cell Resource Centre of Peking Union Medical College before the beginning of the study and spontaneously during the research. Chloroquine, OVA257-264, OVA323^-339, and CY were purchased from Sigma-Aldrich. SB203580, JSH-23, Baf, IPI-549, 1400W 2HCl, and CsA were purchased from Selleck. Anti-CD3 (145^-2C11), anti-CD8 (53-6.7), anti-CD25 (PC-61.5.3) mouse neutralizing antibody, and isotype control (N/A) were purchased form Bio X Cell. Purified anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (RB6-8C5) and rat IgG2bκ isotype control were purchased from BioLegend. The macrophage depletion reagent, clodronate liposomes, was purchased from FormuMax Scientific USA. Ryanodine and CGP37157 were purchased from Abcam.

Generation of HEK293^HA-TDP-43 stable cell lines, expressing HA-tagged WT, E17R, S48A, S48E, M337V, and ΔCR constructs and stable cell lines was previously described [40 (link)]. Mutants A326P and M337P were generated by site-directed mutagenesis as previously described [10 (link)] using the primers listed in S1 Table. Briefly, the HEK293-derived cells Flp-In T-Rex 293 Cell Line (Thermo Fisher Scientific) were stably transfected to express HA-TDP-43 upon induction with tetracycline (1 μg/ml). Cells were grown and maintained in DMEM (Dulbecco’s Modified Eagle’s Medium–High Glucose, Corning) supplemented with 10% FBS (fetal bovine serum) and incubated in a humid atmosphere at 37°C and 5% CO₂. Expression of HA-tagged TDP-43 construct was induced at 30% confluence.

HEK293T cells were purchased from the cell resource center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. They were cultured at 37°C incubator with 5% CO₂ in Dulbecco's modified Eagle's medium (high glucose) (Corning) supplemented with 10% fetal bovine serum (Gibco). The viable cell numbers were counted by trypan blue staining assays. Insect cells, Spodoptera frugiperda Sf9 and High Five cells, were cultured on a shaking table at 26°C and 110 rpm in ESF921 medium (Expression Systems, USA).