Dimethyl sulfoxide (dmso)

Artesunate (MedChemExpress, HY-N0193, China) was dissolved in 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich, D2650, United States) for i.t. injection. Recombinant CCL21 (Abcam, ab201361, United Kingdom), a neutralizing antibody against CCL21 (anti-CCL21, R&D Systems, AF457, United States) and a neutralizing antibody against TREM2 (anti-TREM2, R&D Systems, 1729-T2, United States), was dissolved in normal saline or 10% DMSO for i.t.; delivery and the doses of these reagents were chosen based on previous reports (Hu et al., 2018 (link); Wang et al., 2020a (link); Zhang et al., 2022 (link)). The intrathecal injection was performed under brief anesthesia of sevoflurane (induction, 3.0%; surgery, 1.5%; Maruishi Pharmaceutical Co., Ltd., Japan) and made between the levels of L₅ and L₆ using a 30-G needle (Donnelly et al., 2021 (link)); 5 μl of the reagent was given when the reflexive tail flick was observed.

General caspase inhibitor, Q-VD-OPh, was reconstituted in DMSO (R&D Systems, Minneapolis, MN, USA; OPH001). When used in apoptosis assays, the inhibitor was supplemented to the media at a concentration of 20 μM. 5×10⁵ HT-29 cells were transfected with 1 μg of either the CrmA or DN-FADD plasmid constructs using Effectene (Qiagen, Valencia, CA, USA; 301425). Stable cell lines were generated by selecting with 2 mg/ml Gentamicin (Life Technologies, Carlsbad, CA, USA; 15710-072) for approximately 2 weeks. Gentamicin-resistant cells were maintained under selection pressure until 24 h before treatment.

Recombinant mouse Wnt3a protein (RM Wnt3a, 1324-WN) and DMSO were bought from R&D Systems (Minneapolis, MN, USA) (St. Louis, MO, USA). Drug dosages were determined based on prior studies and our preliminary investigations [23 (link)].

L2 MVECs were seeded in 6-, 24-, 48-, or 96-well plates at the concentration of 5 × 10⁴ cells/mL. After expanding the cells for 24 h, they were washed with medium and stimulated for 24 h with combinations of murine IFN-γ (20 ng/mL, PeproTech, USA), parasite extract (10⁷ infected red blood cells—iRBCs/mL), murine red blood cells extract (10⁷ RBCs/mL), and dexamethasone (100 nM dissolved in DMSO, Sigma). JNK, p38, and ERK inhibitors (SP600125, SB203580, and FR180204, dissolved in DMSO, R&D, UK) were used at 20, 5, and 10 µM, respectively. After stimulation, plates were centrifuged (5 min, 1,200 rpm, RT) and supernatants were collected for ELISA and stored at −20°C. Cytokines were analyzed by ELISA (R&D). Cells were washed with PBS, lysed with RLT buffer with β-mercaptoethanol from RNeasy Mini kit (Qiagen, Belgium), and stored at −80°C for RNA extraction.

The cultured H9C2 cells were seeded in 96-well culture plates, and after transfection and 1 μM ADR treatment, MTT solution (R&D Systems, Minneapolis, MN, USA) was added to each well. The cells continued to incubate for 4 hours in the incubator, then the supernatant of the 96-well plate was carefully discarded. 150 μL dimethyl sulfoxide (DMSO) (R&D Systems, Minneapolis, MN, USA) was added to each well and shaked for 10 minutes to fully melt the crystals. The activity of myocardial cells was detected, and the absorbance value of each well was measured by enzyme-linked immunoassay (Thermo Fisher Scientific, Waltham, MA, USA).

Human recombinant APNs (expressed either in Escherichia coli (E.coli) or in human embryonic kidney (HEK) 293 cells) were purchased from Biovendor (Karasek, Czech Republic). AdipoRon hydrochloride and human recombinant IL-4 (produced in E.coli) were acquired from TOCRIS/R&D Systems (Lille, France) and solubilized respectively in DMSO and Roswell Park Memorial Institute 1,640 medium (RPMI). Antibiotics, DMSO, l-glutamine, trypan blue dye, heat-inactivated fetal calf serum and LPS (from E. coli serotype 0111:B4) were purchased from Sigma (St. Louis, MO, United States). High-molecular-weight poly (I:C) was obtained from InvivoGen (Toulouse, France). Bovine serum albumin and RPMI medium were from Eurobio Biotechnology (Les Ulis, France).