For human IL-6 secretion analysis, iPSC-derived monocytes/macrophages were terminally differentiated in 50 ng/mL human M-CSF for 3-7 days. As a positive control, macrophages from peripheral blood mononuclear cells (PBMCs) were used while mouse embryonic fibroblasts were used as a negative control. PBMCs were isolated from peripheral blood of healthy volunteers using gradient centrifugation with Biocoll Separating Solution (Merck, Germany) for 40 min, 400 g. The healthy donors gave written informed consent according to the local ethical committee at Hannover Medical School. PBMCs were differentiated in RPMI1640 medium with 10% fetal calf serum, 2 mM L-glutamine, 1% penicillin-streptomycin (all Invitrogen, CA, USA) supplemented with 10 ng/mL hM-CSF and 10 ng/mL hIL-3 (Peprotech, Hamburg, Germany) for first 7 days and only 10 ng/mL hM-CSF for next 7 days. IMΦ, murine embryonic fibroblasts and PBMC-derived MΦ were cultivated in 96-well tissue culture plates for 24 h at a density of 6x104 cells/well. After starvation for 24 h in X-Vivo 15 medium, iMΦ were either left unstimulated or were stimulated with 1 µg/mL LPS for another 24 h. Supernatants were collected and analyzed using the human IL-6 human uncoated ELISA Kit (Thermo Fisher, Vienna, Austria) according to the manufacturer´s instructions.
Hm csf
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HM-CSF is a laboratory equipment designed to measure the concentration of colony-stimulating factor (CSF) in cell cultures or biological samples. It utilizes a colorimetric assay to quantify the level of CSF, which is a growth factor that stimulates the production and differentiation of blood cells. The HM-CSF provides researchers with an objective and reliable method to assess the CSF levels in their experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Hil 3, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Lympholyte h, by Cedarlane (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Sting antagonist h151, by Cayman Chemical (2 mentions)
Hgm csf, by Thermo Fisher Scientific (2 mentions)
Versene, by Lonza (1 mentions)
Ficoll gradient, by GE Healthcare (1 mentions)
Rosettesep human monocyte enrichment cocktail, by STEMCELL (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Recombinant porcine il 4, by R&D Systems (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Nunc lab tek chamber slide system, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Biocoll separating solution, by Merck Group (1 mentions)
Defined fbs, by Thermo Fisher Scientific (1 mentions)
19 protocols using hm csf
1
Differentiation and Stimulation of Human iPSC-Derived Monocytes/Macrophages
2
Isolation of Macrophages from NPC1 Carriers
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Blood samples (20 ml) from clinically affected homozygous NPC1 mutation carriers and healthy donors were collected. Negative selection of peripheral blood monocyte-derived macrophages was performed by incubating full blood for 20 min at RT with RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies). An equal volume of washing buffer (D-PBS/2% FBS/1 mM EDTA) was added to each sample and layer of macrophages was separated from red blood cells and plasma by centrifugation on a Ficoll gradient (800 × g for 15 min, GE Healthcare). Potential contamination by red cells was eliminated by incubating cell pellets with ACK lysis buffer (Gibco) for 3 min at RT. Lysis buffer was quenched with 40 ml of washing buffer and cells were centrifuged at 300 × g for 7 min. Cell pellets were resuspended and plated in macrophage complete medium (RPMI 1640/10% FBS/1% PenStrep/1X Pyruvate/1× NEAA) supplemented with 50 ng/ml hM-CSF (Thermo Scientific). After 48 h, 50 ng/ml of fresh hM-CSF was re-added. At 5DIV, media was discarded and adherent cells were washed once in PBS, incubated for 3 min at RT with Versene (Lonza) and scraped in 5 ml macrophage complete medium for further analysis.
3
In Vitro Differentiation and Stimulation of Monocyte-Derived Macrophages
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Mφ cultures were obtained from blood leukocytes, as previously described [21 (link)]. Leukocytes were cultured for 7 days in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 U/mL streptomycin, and 100 μg/mL penicillin (complete RPMI, cRPMI), and with 50 ng/mL of recombinant human M-CSF (hM-CSF) (Thermo Fisher Scientific, Waltham, MA, USA), using Petri dishes, as previously described [21 (link)]. MoMΦ were then harvested, washed, re-suspended in cRPMI and seeded in 12-well plates (Greiner CELLSTAR, Sigma-Aldrich, Saint Louis, MO, USA) (8–10 × 105 live cells per well) or 8-well chamber slides (Nunc Lab-Tek chamber slide system, Sigma-Aldrich) (1 × 105 live cells per well). Cells were incubated at 37 °C 5% CO2 for further 24 h before stimulation. moMΦ were left untreated or stimulated with recombinant porcine IL-10 or TGF-β: culture medium was replaced with fresh cRPMI containing either IL-10 (20 ng/mL) or TGF-β (20 ng/mL) (both R&D Systems, Minneapolis, MN, USA). In defined experiments, culture medium was instead replaced with fresh cRPMI containing LPS (lipopolysaccharide from Escherichia coli 0111:B4; Sigma-Aldrich) (1 μg/mL) or recombinant porcine IL-4 (20 ng/mL) (R&D Systems), which were used as positive controls.
4
Macrophage Culture and Infection Protocol
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Macrophage cultures were obtained from blood leukocytes using Petri dishes and media supplemented with 50 ng/mL of recombinant human M-CSF (hM-CSF) (Thermo Fisher Scientific, Waltham, MA, USA), as previously described [27 (link)]. MoMФ were re-suspended in RPMI-1640 supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (complete RPMI, cRPMI) and seeded in 12-well plates (Greiner CELLSTAR, Sigma-Aldrich, Saint Louis, MO, USA) (7–8 × 105 live cells per well) or 96-well plates (1 × 105 live cells per well). After seeding, cells were incubated at 37 °C 5% CO2 for a further 24 h before infection.
5
Monocyte-Derived Macrophage Culture and Differentiation
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MoMФ cultures were obtained from heparinized blood samples, as we previously published (22 (link)). In brief, leukocytes were cultured in RPMI-1640 supplemented with 10% foetal bovine serum (FBS), antibiotics (100 U/mL penicillin, and 100 μg/mL streptomycin) (complete RPMI, cRPMI), and recombinant human M-CSF (hM-CSF) (Thermo Fisher Scientific, Waltham, MA, USA) (final concentration 50 ng/mL), using Petri dishes (22 (link), 27 (link)). Cells were then seeded in 12-well plates (Greiner CELLSTAR, Sigma-Aldrich, Saint Louis, MO, USA) (1 × 106 live moMФ per well) or 4-well chamber slides (Nunc Lab-Tek chamber slide system, Thermo Fisher Scientific) (3 × 105 live moMФ per well). After seeding, macrophages were cultured in un-supplemented fresh cRPMI at 37°C 5% CO2. 24 h later, moMФ were left untreated or differentiated into moM1, using recombinant porcine IFN-γ (Raybiotech Inc, Norcross, GA, USA) and LPS (lipopolysaccharide from Escherichia coli 0111:B4; Sigma-Aldrich), both at a concentration of 100 ng/mL (27 (link)). 24 h later, both moMФ and moM1 cultures were left untreated or exposed to diverse doses of goat mEVs (0.6, 60 μg) for 24 or 48h. In selected experiments (see 2.5), moMФ were instead exposed to scalar doses of goat milk EVs for 24h: 0.06, 0.6, 6, 60, 600 μg (protein weight).
6
Cell Culture and Monocyte-Derived Macrophage Isolation
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HeLa and RAW264.7 cells were obtained from and authenticated by the American Type Culture Collection. Both cell lines tested negative for mycoplasma contamination by DAPI staining. HeLa cells were grown in DMEM containing L-glutamine and 10% heat-inactivated FCS (MultiCell; Wisent) at 37°C under 5% CO2. RAW264.7 cells were grown in RPMI-1640 medium containing L-glutamine and 10% heat-inactivated FCS at 37°C under 5% CO2.
To obtain nonpolarized human monocyte-derived macrophages, peripheral blood mononuclear cells were isolated from the blood of healthy donors by density-gradient separation with Lympholyte-H (Cedarlane). Human monocytes were then separated by adherence and incubated in RPMI-1640 containing L-glutamine, 10% heat-inactivated FCS, 100 U · ml−1 penicillin, 100 µg · ml−1 streptomycin, 250 ng · ml−1 amphotericin B, and 25 ng · ml−1 hM-CSF (PeproTech) for 5–7 d, at 37°C under 5% CO2, before experimentation.
To obtain nonpolarized human monocyte-derived macrophages, peripheral blood mononuclear cells were isolated from the blood of healthy donors by density-gradient separation with Lympholyte-H (Cedarlane). Human monocytes were then separated by adherence and incubated in RPMI-1640 containing L-glutamine, 10% heat-inactivated FCS, 100 U · ml−1 penicillin, 100 µg · ml−1 streptomycin, 250 ng · ml−1 amphotericin B, and 25 ng · ml−1 hM-CSF (PeproTech) for 5–7 d, at 37°C under 5% CO2, before experimentation.
7
Characterizing Bone Marrow-Derived Macrophages
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Bone marrow cells were isolated from femurs and tibias of mice and cultured for 7 days in RPMI medium including 10% FBS and 1% Penicillin-Streptomycin-Glutamine with 50ng/ml hM-CSF (Peprotech). Cells were plated 350,000/700μl and stimulated with LPS (100ng/ml), Poly I:C (10ng/ml), CpG (1μg/ml) or cGAMP (5μg/ml unless otherwise stated). STING antagonist H151 (Cayman chemicals) was added 30 minutes before stimulation.
8
iPSC-Derived Microglial Characterization
9
Isolation and Differentiation of Primary Human Monocytes
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Primary human monocytes were isolated from heparinized blood of donors. Peripheral blood mononuclear cells were first isolated using Lympholyte-H (Cedarlane), resuspended in RPMI, and seeded onto tissue culture-treated plastic dishes for 20 minutes to select adherent cells. Nonadherent cells were removed by washing with RPMI medium (Wisent Bioproducts, catalog 350-000-CL). Adherent cells were then incubated in RPMI with L-glutamine containing 10% heat-inactivated serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10 ng/mL hM-CSF (PeproTech, catalog 300-25) for 7 days.
10
Characterizing Bone Marrow-Derived Macrophages
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