BMDCs were generated as previously described36–40 with some modifications. Briefly, bone marrow cells were harvested from the femur and tibia of the hind legs. Next, the cells were depleted of red blood cells (RBCs) with RBC lysis buffer (Lonza, Walkersville, MD, USA). The cells were cultured for 6 days in RPMI 1640 (Lonza) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 10 000 units/ml penicillin (Gibco), 10 ng/ml rmGM-CSF (JW CreaGene, Daegu, Korea) and 10 ng/ml rmIL-4 (JW CreaGene, Daegu, Korea). The culture medium was replaced every 2 days. After 7 days, DCs were incubated for 24 h with 10 ng/ml rmTNF-α (PeproTech, Rocky Hill, NJ, USA) or 1 μg/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) to generate smDCs or fully mDCs, respectively. At the same time, 1 μg/ml rhSNCG (Acris, San Diego, CA, USA) was co-administered to differentiate the DCs to investigate the effect of SNCG. The purity of the BMDC population was assessed by flow cytometry after CD11c labeling (>90% for CD11c+ cells).
Rmtnf α
Manufactured by Thermo Fisher Scientific
Sourced in United States
RmTNF-α is a recombinant protein that corresponds to the mouse tumor necrosis factor alpha (TNF-α) molecule. It is a laboratory reagent used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rmgm csf, by Thermo Fisher Scientific (3 mentions)
Rmil 4, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Rbc lysis buffer, by Lonza (1 mentions)
Boyden chamber, by Merck Group (1 mentions)
Rhil 22, by R&D Systems (1 mentions)
Igg2a, by Genentech (1 mentions)
Ampicillin sodium salt, by Merck Group (1 mentions)
Endotoxin free water, by Merck Group (1 mentions)
Gsk 872, by Merck Group (1 mentions)
Ap21967 ac heterodimerizer, by Takara Bio (1 mentions)
Z vad fmk, by Enzo Life Sciences (1 mentions)
8 protocols using rmtnf α
1
Generating Bone Marrow-Derived Dendritic Cells
2
Neutralizing Antibodies in IL-22 Study
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IL-22 neutralizing antibodies and IgG2a control were provided by Genentech (San Francisco, CA). rmIL-22 and rmTNF-α were from Peprotech (London, UK), rmIL-1a and rhIL-22 from R&D Systems (Minneapolis, MN), ELISA kits from Peprotech, R&D or Antigenix America and Boyden chambers from Millipore (Billerica, MA).
3
Murine TNFα Antibody Production and Antibiotic Dosage
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Rabbit anti-murine TNFα serum was raised by Cambridge Research Biochemicals (Cambridge, UK) via immunization with recombinant murine TNFα (rmTNFα, Peprotech, London, UK). Five micrograms of rmTNFα was administered subcutaneously in Freund's complete adjuvant, followed by 5 and 25 μg booster doses in Freund's incomplete adjuvant after 28 and 56 days, respectively. IgG was purified from serum using Protein A Plus spin kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions, to a final concentration of 2 mg/mL. Samples were sterile filtered, and stored at –20°C until use. Purity of IgG was assessed by SDS-PAGE after Coomassie staining (bands of 50 and 23 kDa, corresponding to heavy and light chains, respectively), whereas specificity of anti-TNFα IgG was determined by western blot analysis. Rabbit IgG antibodies (Thermo Scientific) were used as control.
Ampicillin sodium salt and ciprofloxacin hydrochloride powders (Sigma Aldrich, Gillingham, UK) were resuspended in endotoxin-free water (Sigma Aldrich) to obtain the desired concentrations of antimicrobials and sterile filtered freshly before injections. The maximum upper dosage indicated for veterinary treatment of small rodent infections was chosen for our study (150 mg/kg in the case of ampicillin treatment and 20 mg/kg in the case of ciprofloxacin treatments).
Ampicillin sodium salt and ciprofloxacin hydrochloride powders (Sigma Aldrich, Gillingham, UK) were resuspended in endotoxin-free water (Sigma Aldrich) to obtain the desired concentrations of antimicrobials and sterile filtered freshly before injections. The maximum upper dosage indicated for veterinary treatment of small rodent infections was chosen for our study (150 mg/kg in the case of ampicillin treatment and 20 mg/kg in the case of ciprofloxacin treatments).
4
Peritoneal Lavage for Immune Cell Analysis
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Mice received a single intraperitoneal dose of 500 ng rm‐TNF‐α (PeproTech). After overnight, peritoneal lavage was performed by injecting 5mL PBS containing 1 mM EDTA into PC with gentle rubbing, volume was recovered afterward and prepared for flow cytometric analysis.
5
Immunomodulatory Effects of hucMSCs-Derived Exosomes on Dendritic Cells
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We observed whether hucMSCs-Exo affected the activation and maturation of DCs and the release of cytokines. DCs were obtained from the BM of normal 4-to 6-week-old female C57BL/6 mice according to a previously described protocol [28] . BM cell pellets were resuspended in RPMI-1640 medium (GIBCO) supplemented with 10% FBS (GIBCO, Australia), 1% penicillin-streptomycin, 20 ng/ml rmGM-CSF (PeproTech, USA), and 20 ng/ml rmIL-4 (PeproTech, USA) at 1 × 10 6 cells/ml and transferred into a 6-well plate. On day 3 of culture, non-adherent cells were gently removed, and fresh culture medium was added. Fresh culture medium was added every 2 days thereafter to induce DC differentiation. For DC maturation, on day 7, non-adherent cells were collected, and DCs were washed, resuspended at 2 × 10 5 cells/ml in culture medium with 10 ng/ml rmTNF-α (PeproTech, USA), and seeded in a 24-well plate, with or without hucMSCs-Exo (2.5 µg/ml) co-cultured for another 24 h. The different groups of cells were collected to detect the phenotype of DCs by flow cytometry, and the cell supernatants were collected and centrifuged at 1000 × g for 20 minutes at 4 °C. Then, the supernatants were stored at -80 °C and utilized for IL-23p40 ELISAs within one month.
6
Modulating Dendritic Cell Function with hucMSCs-Exo
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We observed whether hucMSCs-Exo affected the activation and maturation of DCs and the release of cytokines. DCs were obtained from the BM of normal 4-to 6-week-old female C57BL/6 mice according to a previously described protocol [28] . BM cell pellets were resuspended in RPMI-1640 medium (GIBCO) supplemented with 10% FBS (GIBCO, Australia), 1% penicillin-streptomycin, 20 ng/ml rmGM-CSF (PeproTech, USA), and 20 ng/ml rmIL-4 (PeproTech, USA) at 1 × 10 6 cells/ml and transferred into a 6-well plate. On day 3 of culture, non-adherent cells were gently removed, and fresh culture medium was added.
Fresh culture medium was added every 2 days thereafter to induce DC differentiation. For DC maturation, on day 7, non-adherent cells were collected, and DCs were washed, resuspended at 2 × 10 5 cells/ml in culture medium with 10 ng/ml rmTNF-α (PeproTech, USA), and seeded in a 24-well plate, with or without hucMSCs-Exo (2.5 µg/ml) co-cultured for another 24 h. The different groups of cells were collected to detect the phenotype of DCs by ow cytometry, and the cell supernatants were collected and centrifuged at 1000 × g for 20 minutes at 4 °C. Then, the supernatants were stored at - 80 °C and utilized for IL-23p40
ELISAs within one month.
Fresh culture medium was added every 2 days thereafter to induce DC differentiation. For DC maturation, on day 7, non-adherent cells were collected, and DCs were washed, resuspended at 2 × 10 5 cells/ml in culture medium with 10 ng/ml rmTNF-α (PeproTech, USA), and seeded in a 24-well plate, with or without hucMSCs-Exo (2.5 µg/ml) co-cultured for another 24 h. The different groups of cells were collected to detect the phenotype of DCs by ow cytometry, and the cell supernatants were collected and centrifuged at 1000 × g for 20 minutes at 4 °C. Then, the supernatants were stored at - 80 °C and utilized for IL-23p40
ELISAs within one month.
7
Apoptosis and Necroptosis Induction
8
Generation and Adenoviral Transduction of Murine Bone Marrow-Derived Dendritic Cells
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Bone marrow-derived DCs were prepared as described (Lutz et al., 1999) . DCs were generated from C57BL/6 mouse bone marrow (BM) progenitor cells as follows: cells from bone marrow were flushed out from hind legs with a syringe, passed through a nylon mesh and collected. Red blood cells were lysed with lysis buffer containing ammonium chloride. Cells were then cultured in RPMI1640 containing 10% FBS (Life Technologies), rmGM-CSF (50 ng/mL, PeproTech) and rmIL-4 (25 ng/mL, PeproTech). On the 3th day, half of the culture supernatant was collected and centrifuged and the cell pellet was resuspended in RPMI1640 containing the cytokines. The cells were then returned to the original plate. DCs were matured in the presence of rmTNF-a (100 ng/mL, PeproTech) on the 6th day. Two days later, the DCs were then pulsed with adenovirus at different multiplicity of infection (MOI) for 48h. Percentage and maturation status of the harvested DCs were examined by flow cytometry.
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