Macrophage colony stimulating factor m csf

Lomerizine was purchased from Absin Bioscience Inc. (Shanghai, China). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, United States). Dexamethasone was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Hematoxylin and eosin (H&E) staining kit was purchased from Abcam (Waltham, Boston, United States). Dulbecco’s Modified Eagle Medium (DMEM) and macrophage colony-stimulating factor (M-CSF) was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Penicillin-streptomycin solution (Pen Strep) was purchased from Yeasen Biotechnology Co., Ltd. (Shanghai, China). Fetal Bovine Serum (FBS) was purchased from Cegrogen Biotech (Wupperweg, Germany).
Primers were synthesized by Hua Gene Biotech Co., Ltd. (Shanghai, China). The primary antibody against IκB-α was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Primary antibodies against iNOS, phospho-NF-κB p65 (Ser536) Rabbit mAb, NF-kappaB p65 Rabbit mAb, phospho-p38 MAPK (Thr180/Tyr182) XP Rabbit mAb, p38 MAPK XP Rabbit mAb, phospho-SAPK/JNK (Thr183/Tyr185) Rabbit mAb, SAPK/JNK Antibody, Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) Antibody, p44/42 MAPK (ERK1/2) Rabbit mAb, and β-Actin Rabbit Antibody were purchased from Cell Signaling Technology (Danvers, MA, United States).

Cultures were prepared from post-natal C57BL/6 mouse pups at 2 days of age. Whole brains were trypsin digested and made into a cell suspension. Cells were seeded in flasks pre-coated with 0.1 mg/ml poly-D-lysine (Sigma-Aldrich) and maintained in DMEM supplemented with 10% heat-inactivated FBS (Gibco) and 1% Pen-Strep (Gibco). Medium was supplemented with 5 ng/ml Macrophage Colony Stimulating factor (M-CSF) (Thermo Fisher Scientific) diluted in PBS supplemented with 0.1% sterile filtered BSA (Sigma-Aldrich). At DIV10 microglia were isolated by orbital shaking at 150 RPM for 1 h and the supernatant was seeded in 12-well plates with 300,000 cells per well. Experiments were performed on the subsequent day. PHF-tau (paired helical filaments) [69 (link)] was added to microglia at a concentration of 1 μg/ml as determined by total tau ELISA. ScFvMC1 was added at a concentration of 10 μg/ml. To allow for immune complex formation, PHF-tau and scFvMC1 were mixed in medium and pre-incubated at 37 °C for 30–45 min prior to addition to cells. Mixing was performed two times during incubation by repeated manual pipetting. The 2 h incubation was performed in medium without serum. All experiments were performed in triplicate, with each treatment group in quadruplicate. The amount of PHF-tau in medium at the end of the experiments was assessed using the same low-tau ELISA previously described.

For BMDCs, bone marrow cells were harvested from mice into RPMI 1640 (Sigma-Aldrich) supplemented with 5% heat-inactivated FCS (Hyclone, Logon, UT), 100 μg /mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich), 2 mM L-Glutamine (Sigma-Aldrich), 50 μM β-mercaptoethanol (Gibco) and 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (eBioscience). For BMDMs, BM cells were harvested from mice into modified Eagle′s Medium (DMEM)–high glucose (Sigma-Aldrich) supplemented with 5% heat-inactivated FCS (Hyclone, Logon, UT), 100 μg/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich), Eagle′s minimum essential medium (MEM) non-essential amino acid solution (Sigma-Aldrich), and 0.1 mg/ml macrophage colony-stimulating factor (M-CSF) (eBioscience). BMDMs and BMDCs on day 8 were collected at 1X10⁶/ml and incubated with 10 μg/ml VLP recombinant protein for overnight at 37°C. ES at a final concentration 50 μg/ml and lipopolysaccharide (LPS) at final concentration 0.1 μg/ml were used as positive controls, and untreated cells were used as a negative control. After 24 hours, the supernatants were harvested separately and stored at -20°C until analysed for cytokine content by Legendplex or CBA according to the manufacturer’s instructions.

Cell culture reagents used were Penicillin-Streptomycin, RPMI 1640 obtained from Cambrex (Belgium) and fetal bovine serum from Life Technologies Ltd (UK). The TLR ligands used were chloroform extracted Escherichia coli (E.coli) lipopolysaccharide (LPS) and resiquimod (R-848) from Invivogen (USA). Flagellin (purified) and Pam₃cys-ser(lys)₄.3HCl (Pam3) were from Alexis (UK). Mianserin hydrochloride was purchased from Sequoia Research Products (Pangbourne, UK). The structural derivatives of mianserin were synthesized by Oxygen Healthcare Research Pvt. Ltd. (Ahmedabad, India), the synthesis steps are provided in the Supplemental Information. Macrophage colony stimulating factor (M-CSF) was purchased from eBioscience (USA). Dimethyl sulfoxide (DMSO) and Hank's balanced salt solution (HBSS) without calium chloride and magnesium sulfate were purchased from (Sigma, UK). Percoll Plus was purchased from GE Healthcare (Bucks, UK), PBS citrate from Paris Anticorps (France) and lympholyte-H from CedarLane (Ontario, Canada).

Institutional approval from the local research ethical committees (Internal Review and the Ethics Boards of the Tongji Hospital, Tongji University) was obtained prior to conducting the study. Human peripheral blood monocyte-derived macrophages were generated as previously described [6 (link)]. Briefly, human peripheral blood mononuclear cells (PBMC) from healthy blood donors from the blood bank of the Tongji Hospital of Tongji University were isolated from buffy coats by Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) density centrifugation. PBMCs were allowed to adhere to culture flasks for 1 h at 37°C in DMEM supplemented with 1% human serum, after which the nonadherent cells were removed by vigorous washing with PBS. Adherent cells were cultured in 20 ml DMEM (10% FCS) supplemented with 50 ng/ml macrophage colony stimulating factor (M-CSF) (eBioscience, San Diego, CA, USA) for 7 days to allow differentiation to macrophages.

Human monocytes or natural killer (NK) cells were isolated from buffy coats (Sanquin, Amsterdam, the Netherlands) from healthy blood donors <24 h after blood collection. All donors gave informed consent. Whole blood was diluted 1 : 1 in PBS and loaded on Lymphoprep (Nyegaard, Oslo, Norway), whereafter cells were separated by density centrifugation. Peripheral blood mononuclear cells (PBMC) were extracted from the interphase of the Lymphoprep gradient and washed three times with PBS supplemented with autologous serum. Either CD14+ monocytes or NK cells were isolated from the PBMC fraction with cell separation beads (positive selection for CD14+ monocytes, negative selection for NK cells) (Miltenyi Biotech, Leiden, the Netherlands), according to the manufacturer's protocol. Isolated cells were washed and resuspended in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 200 μML glutamine (hereafter referred to as complete RPMI). CD14+ monocytes were cultured in complete RPMI with 50 ng/ml macrophage colony-stimulating factor (M-CSF) (eBioscience, San Diego, CA) for eight days.

Mice were killed, and the peritoneal cavity was lavaged with two washes of 5 ml of RPMI (containing 1% penicillin-streptomycin and 10% FBS). The recovered media was  centrifuged (432 × g for 10 min). For RNA analysis, the resultant pellet of peritoneal exudate cells was lysed immediately, and RNA extraction was carried out (as described below). For photomultiplier tube recordings, the pellet was re-suspended in RPMI, and cells were plated out into a 35-mm dish. After 2-h incubation at 37 °C, non-adherent cells were removed via three washes in warmed RPMI. For bone marrow-derived macrophages, mice were killed and the femur and tibia were removed. The bone marrow was flushed out with DMEM (containing 1% penicillin-streptomycin and 10% FBS), and the effluent was centrifuged (432 × g for 10 min). The pelleted cells were re-suspended in media containing 50 ng/ml macrophage colony-stimulating factor (M-CSF) (eBioscience, San Diego, CA, USA). The media were replaced after 3 days, and on day 6, the bone marrow-derived macrophages were scraped from the flask and lysed to extract protein for Western blot analysis (described below).