Mip 1α

RAW264.7 cells and HEK293 cells are obtained from ATCC, and maintained in DMEM (Dulbecco′s modified Eagle′s medium) supplemented with 10% fetal bovine serum (FBS) from Gibico. BSA (bovine serum albumin) and anti-BSA antibody (α-BSA) are obtained from Invitrogen and MP Biomedicals, respectively. Dimethyl sulfoxide (DMSO) is obtained from Sigma-Aldrich. Rolipram and Roflumilast are obtained from Cayman Chemical and APExBIO, respectively. 6-Bnz-cAMP (PKA agonist) and 8-pCPT-2′-O-Me-cAMP (Epac agonist) are obtained from Biolog Life Science Institute. PKA inhibitor H-89 is obtained from Beyotime Biotechnology. ELISA kits for measuring cAMP, TNF-α, MIP-1α, MIP-1β, MIP-2 and KC are all obtained from R&D Systems. ELISA kit for measuring mouse albumin is obtained from Bethyl Laboratories.

Analysis of growth factors contained in the skin around the magnet-implanted ulcers was performed 7 days after ulcer production. To analyze cytokines in tissues after treatment of the magnet-implanted ulcers, the skin around the ulcer treated with trafermin or mixed cell sheets was collected the following day. The skin around the flesh of the ulcers just after the 7-day implantation of the magnet and the skin around the untreated ulcers were collected as controls. Protein was extracted from the tissues using lysis buffer, and an ELISA was performed to measure VEGF, PDGF-BB, TNF-α, IL-6 and MIP-1α production (R&D Systems) according to the manufacturer’s protocol.

Supernatants from brain homogenates were prepared for cytokine and chemokine measurement by enzyme-linked immunosorbent assay (ELISA) after 3 weeks subsequent to intracerebral M. tuberculosis infection. The chemokines MCP-1, MIP-1α, and RANTES; and the cytokines IFN-γ, IL-2, IL-6, IL-12p70, and TNF (R&D Systems, Germany) were measured using commercially available ELISA reagents according to the manufacturer’s instructions. Chemokine and cytokine concentrations were measured by absorbance using a Versamax Microplate Reader (Molecular Devices, LLC, CA, USA) with SoftMax software.

After LDLR^–/– mice were fasted for 6 h, blood samples were collected from heart. The samples were centrifuged at 500 g for 20 min at 4°C to separate plasma. Plasma TC and TG contents, HDL-C and LDL-C levels were detected with kits from BioSino Bio-technology and Science Inc. (Beijing, China). Plasma AST, ALT and ICAM-1 levels were measured by enzyme-linked immunosorbent assay using an insulin ultrasensitive ELISA kit (R&D, Minneapolis, MN, United States), according the instruction of the manufacture. The color absorbance at 450 nm was measured using a Bio-Rad microplate reader.
Plasma cytokines were determined by BD Cytometric Bead Array with a flow cytometer (FACS Calibur, BD, Franklin Lakes, NJ, United States) at the end of experiments as described previously (Zhang et al., 2014 (link); Mao et al., 2015 (link)). The following cytokines were determined in this study using their corresponding antibody: IL-1α, IL-1β, IL-6, IL-10, TNF-α, MCP-1, and MIP-1 α (R&D Systems, Minneapolis, MN, United States).

At 24 h after the mice were administered with ZY or NS or at 6 h after the KCs were treated with ZY or CM, the cytokine and chemokine levels in the perfusate or KC culture supernatants were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits. Mouse TNF-α, IL-1β, IL-6, HMGB-1, MIP-1α, MIP-2, and MCP-1 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Optical density was measured on an ELISA plate scanner (CA94089, Molecular Devices, Sunnyvale, Canada). All experiments were performed according to manufacturer's instructions [28 (link)].

GYF-21 was isolated from Chinese agarwood and the isolation procedure was described primarily (Huo et al., 2015 (link)). GYF-21 was dissolved at a concentration of 25 mM in dimethyl sulfoxide (DMSO). Cell Counting Kit-8 (CCK-8) was purchased from Beyotime, Co. (Shanghai, China). Lipopolysaccharide (LPS) from Escherichia coli O55: B5 was purchased from Sigma Chemicals, Co. (St. Louis, MO, United States). ELISA kits for determining TNF-α,IL-6, IL-1β, MCP-1, MIP-1α, IFN-γ, and IgG were purchased from R&D Systems (Minneapolis, MN, United States). The antibodies to p65, I-κB, p38, ERK1/2, JNK, STAT1, STAT3, and their phosphorylated forms were purchased from Cell signaling Technology (Beverly, MA, United States). Monoclonal antibodies (mAbs) conjugated to APC, APC, PE, FITC, PE, FITC, FITC, PE, PE-Cy7, Alexa Fluor 647, FITC, and PE (specific for Ly-6G, CD11c, CD11b, CD62L, CD69, CD25, CD80, CD86, CD4, CD8, IFN-γ, and IL-17A), and purified monoclonal antibodies (specific for CD3e, CD28, IFN-γ, and IL-4) were obtained from Becton Dickinson (San Diego, CA, United States). Magnetic bead isolation kit for mouse CD4⁺ T cells, naive CD4⁺ T cells and CD8⁺ T cells were purchased from Miltenyl Biotec (Bergisch Gladbach, Germany). Recombinant mouse GM-CSF, IL-4, IL-12, IFN-γ, IL-6, and TGF-β were purchased from PeproTech (Rocky Hill, NJ, United States).

Based on the differential usage of co-receptors (CCR5 and CXCR4), HIV isolates have been referred to as R5-, X4-, or dual-tropic strains [32 (link)]. The HIV R5-tropic strains (Bal, Jago, and YU2) were obtained from the AIDS Research and Reference Reagent Program of the National Institute of Health (NIH, Bethesda, Rockville, MD, USA). Imiquimod, a synthetic TLR7 ligand, was purchased from InvivoGen (San Diego, CA, USA). MIP-1α, MIP-1β, and RANTES antibodies were purchased from R&D System (R&D system Inc., Minneapolis, MN, USA). Mouse IgG was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-Mouse IgG (horseradish peroxidase (HRP)-linked) antibody, and anti-rabbit IgG (HRP-linked) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse antibody against HIV-1 p24 was purchased from Abcam (Abcam, Cambridge, UK). All antibodies and reagents for flow cytometry assay were purchased from BD Bioscience (BD Bioscience, San Jose, CA, USA). Trichloroacetic acid (TCA) and acetone were purchased from Sigma-Aldrich.