Rpmi 1640 cell medium

Imatinib-sensitive K562 cells were kindly given by Institute of Clinical Pharmacology (Central South University), imatinib-resistant K562/G01 cells were kindly given by Tianjin Institute of Hematology. K562 and K562/G01 cells were cultured in RPMI 1640 cell medium (Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Gibco, South America) and 1% penicillin-streptomycin (Gibco, United States) mixture maintained at 37°C in a cell incubator with a humidified atmosphere containing 5% CO2. K562/G01 was cultured with 4 μM imatinib to keep its resistance, and removed imatinib treatment 1 week before experiment.
For hsa_circ_0058493 downregulation, K562/G01 cells were infected with lentivirus vector (GV493) carrying short hairpin RNA (shRNA) against hsa_circ_0058493 (sh-circ_0058493) and its negative control (sh-NC) (Genechem, Shanghai, China) following the manufacturer’s instructions. Puromycin (biosharp, China) were added into cells after transfection to screen successfully transfected cells. The sequence of sh-circ_0058493: sh-circ_0058493-1, CTC​ACC​AGG​GTT​CAG​CCG​T; sh-circ_0058493-2, TCA​CCA​GGG​TTC​AGC​CGT​C; sh-circ_0058493-3, TTT​ATT​CTC​ACC​AGG​GTT​C, and the one with the best knockdown efficiency would be chosen to conduct the subsequent experiments.

Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA anticoagulated blood. Blood and phosphate-buffered saline supplemented with 0.5% bovine serum albumin (PBS/BSA) were mixed at a 1:1 ratio and 35 ml were layered onto 15 ml Ficoll-Paque PLUS gradient (GE Healthcare). PBMCs were isolated according to the manufacturer’s protocol and stored in liquid nitrogen until the analysis.
For analysis batches of 20–35 samples were thawed and washed in RPMI 1640 cell medium (Gibco). In each batch, a mix of samples from healthy controls and MS/NMOSD patients were included. Cells were stained with antibodies against CD14 (DRFZ, clone TM1), CD169 (SIGLEC1, clone 7–239, BioLegend Cat# 346004, RRID:AB_2189029) and a dead cell stain (eBioscience Fixable Viability Dye eFluor 780). For each batch, a fluorescence minus one (FMO) control for SIGLEC1 was measured and FMO measurements remained stable over multiple batches. The samples were acquired on a FACSCanto cytometer (BD Biosciences) and all cytometry experiments were performed according to published standards^{26 (link)}. Using the FlowJo software (Version 10.4.1 for Mac, FlowJo LLC), we identified monocytes and excluded doublets based on scatter parameters. We then gated on CD14^high, living cells and analysed the median fluorescence intensity (MFI) for SIGLEC1 (Fig. 1a).

Reduced glutathione (GSH), digitonin, dimethyl sulfoxide (DMSO), 2,2,2-trichloroacetic acid (TCA), 2,4-dinitrophenylhydrazine (DNPH), 5,5’-dithiobionitrobenzoic acid (DTNB) and diisopropyl fluorophosphate (DFP) were purchased from Sigma Chemical Company (St. Louis, MO). DMEM, KSFM and growth factors, and RPMI 1640 cell medium, penicillin/streptomycin solution, and geneticin (G418) and KB plus DNA ladder, Celltracker blue (7-amino-4-chloromethylcoumarin or CMAC), 10kD spin columns, and EnzChek Caspase-3 assay kit were purchased from Invitrogen (Grand Island, NY). BCA kit and the anti-DYKDDDDK (anti-FLAG) antibody (PA1-984B) were purchased from Pierce (Rockford, IL). Celltiter 96 AQueous One MTS kit, described as the MTS viability assay in experiments, was purchased from Promega (Madison, WI) and contained CellTiter96 Aqueous One Solution composed of a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium, inner salt (MTS) and an electron coupling reagent (phenazine methosulfate). The Apoptotic DNA ladder kit was purchased from Roche (Indianapolis, IN). All chemicals used for the synthesis of prodrugs were purchased from Sigma-Aldrich (St. Louis, MO), TCI (Portland, OR), Acros Organics (Thermo Fisher Scientific, New Jersey) and Lancaster (Ward Hill, MA) and used without further purification.

Single-use plastic equipment was supplied by NUNC (Thermo Fisher Scientific, Roskilde, Denmark). RPMI 1640 cell medium, fetal calf serum (FCS, inactivated), and other cell media were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). The following monoclonal antibodies (mAb) were used in the flow cytometric study: anti-human CD14 (IgG2bκ PercPmAb, clone: MφP9) to mark peripheral blood monocytes; anti-human CD64 (IgG1κ FITCmAb, clone:10.1), anti-human CD32 (IgG2bκ FITCmAb, clone: FLI8.26), and anti-human CD16 (IgG1k FITCmAb, clone: 3G8) to analyze the presence of Fcγ receptors on these monocytes; anti-human CD35 (IgG1k PEmAb, clone:E11), anti-human CD11b (IgG1κ PEmAb, clone: ICRF44), and anti-human CD11c (IgG1k PEmAb, clone: B-ly6) to analyze the presence of complement receptors on these cells (all antibodies were purchased from BD Bioscience, PharMingen, Heidelberg, Germany). Appropriate surface isotype controls were implemented.

Cells from each group were plated evenly on a 24-well tissue culture dishes and incubated overnight at 37° C, 5% CO₂. Each dose of SKI-II inhibitor (Santa Cruz Biotechnology cas 312636-16-1) was prepared by serial dilution in complete RPMI 1640 Cell Medium (Thermo Fisher cat # 11875093). The SKI-II treated volumes of cell medium were sterilized on 0.2 μm filters and heated to 37° C. Wells containing adhered cells in the tissue culture dishes were aspirated and washed twice with 1x PBS. One mL of SKI-II treated medium was added to each well. Real-time phase object confluence was monitored over time using an Incucyte ZOOM^® Live-Cell Analysis System at 37° C, 5% CO₂ and quantitative data were analyzed using the Incucyte ZOOM^® data analysis software to generate cell growth curves. Proliferation rates were determined by calculating the mean slope of the linear-like growth phases of 3–4 biological replicates per group.