Human monocyte-derived macrophages (HMDM) were isolated from the peripheral blood of healthy donors as previously described. Briefly, blood was layered at a ratio of 2:1 (blood/Ficoll medium) on Ficoll Histopaque-1077 (Sigma) in 15 ml centrifuge tubes and spun for 30 min at 2000 rpm in an Allegra X-22R centrifuge (Beckman Coulter). The layer containing the peripheral blood mononuclear cells was collected and then resuspended in 15 ml of PBS, and recentrifuged for 10 min at 1000 rpm. After two washes in PBS, cells were resuspended in DMEM containing 10% FBS, L-Glutamine and 100 units ml−1 penicillin and 100 mg ml−1 streptomycin on 12 mm diameter coverslips in 24-well plates. Non-adherent cells were removed after 4 h. The cells were subsequently cultured in cell culture medium containing 50 ng ml−1 granulocyte macrophage colony stimulating factor (GM-CSF) (Sigma Aldrich) in an atmosphere containing 5% CO2. Cultures were fed daily, and infection experiments were performed 10 days after the peripheral blood was collected. Infections were performed with MOI of 100:1:1 (bacteria/neutrophil/macrophage) ratio.
Granulocyte macrophage colony stimulating factor gm csf
Manufactured by Merck Group
Sourced in United States, Australia
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the production and differentiation of granulocytes and macrophages from bone marrow progenitor cells. It plays a crucial role in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Ficoll histopaque 1077, by Merck Group (1 mentions)
Allegra x 22r centrifuge, by Beckman Coulter (1 mentions)
α thioglycerol, by Merck Group (1 mentions)
Hmc 1, by American Type Culture Collection (1 mentions)
Ti45 rotor, by Beckman Coulter (1 mentions)
Pen strep, by Merck Group (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Macrophage colony stimulating factor m csf, by Merck Group (1 mentions)
Heat inactivated, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Merck Group (1 mentions)
Titanium dioxide nanoparticles tio2, by Merck Group (1 mentions)
1 3 diphenylisobenzofuran dpbf, by Merck Group (1 mentions)
Rpmi 1640, by InvivoGen (1 mentions)
Fitc labeled cpg, by InvivoGen (1 mentions)
7 protocols using granulocyte macrophage colony stimulating factor gm csf
1
Isolation of Human Monocyte-Derived Macrophages
2
Culturing Human Mast and Erythroleukemic Cells
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The human mast cell line HMC-1 (Dr. Joseph Butterfield, Mayo Clinic, Rochester, MN, USA) and the erythroleukemic cell line TF-1 (ATCC: CRL-2003) were cultured in a 37°C humidified incubator with 5% CO2. HMC-1 was cultured in Iscove's Modified Dulbecco's Medium (IMDM), while TF-1 was cultured in RPMI-1640. Both media were supplemented with 10% foetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L -glutamine. In addition, the medium for HMC-1 also contained 1.2 mM α-thioglycerol and the medium for TF-1 was supplemented with 5 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) (all reagents were from Sigma-Aldrich, St Louis, MO, USA).
To eliminate serum exosomes, the FBS was ultracentrifuged (Ti45 rotor, Beckman Coulter, Brea, CA, USA) for 18 hours at 120,000 × g (average) prior to use in cultures.
The cell viability was calculated using the trypan blue dye method.
To eliminate serum exosomes, the FBS was ultracentrifuged (Ti45 rotor, Beckman Coulter, Brea, CA, USA) for 18 hours at 120,000 × g (average) prior to use in cultures.
The cell viability was calculated using the trypan blue dye method.
3
PBMC Isolation and Cultivation
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Human PBMCs were isolated from buffy coat (Hungarian National Blood Transfusion Service) from healthy donors by Ficoll Paque Plus (GE Healthcare) gradient centrifugation as described previously (43 (link)). PBMCs were washed with ice- cold phosphate-buffered saline (PBS) ( 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4[ pH 7.4]). Isolated PBMCs were suspended in RPMI 1640 supplemented with 1% 100× penicillin-streptomycin solution (Pen-Strep; Sigma-Aldrich) and the concentration of cells had been adjusted to 2 × 107/ml; 2 × 107, 107, or 5 × 105 PBMCs were transferred to 12-, 24-, or 96- well plates, respectively. After 1.5 h of incubation (5% CO2, 37°C, 100% relative humidity), floating cells were removed by changing the culture medium to AIM-V cell culturing medium (Gibco) supplemented with 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) ( Sigma-Aldrich) every other day for 7 days.
4
Isolation and Differentiation of Human Monocyte-Derived Macrophages
5
Photodynamic Therapy with TiO2 Nanoparticles
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Titanium dioxide (TiO2) nanoparticles and Chlorin e6 (Ce6) were purchased from Sigma-Aldrich. CpG oligonucleotide (ODN) and FITC-labeled CpG were purchased from Invivogen. 1,3-diphenylisobenzofuran (DPBF) and 2′,7′-dichlorofluorescein diacetate (DCF-DA) were obtained from Sigma-Aldrich. 4ʹ,6-diamidino-2-phenylindole was obtained from Beyotime Biotechnology. Anti-PD-L1 (aPd-L1), anti-CD11c and anti-CD86 antibodies were purchased from Invivogen. Tumor necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were obtained from Sigma-Aldrich. For cell culture, RPMI-1640, DMEM, trypsin-EDTA, and fetal bovine serum (FBS) were purchased from Invivogen. All ELISA Kit was purchased from Sigma-Aldrich. All chemicals were of analytical grade and no further purification was required.
6
Generation of Tolerogenic Monocyte-Derived DCs
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Monocyte-derived DCs (MoDCs) were generated from CD14+ monocytes from PBMCs. Briefly, monocytes were purified by positive selection with an anti-CD14 antibody coupled to magnetic beads (Miltenyi Biotec). The isolated monocytes were treated with mitomycin C (50 μg/mL, Sigma-Aldrich) for 20 minutes at 37°C. To obtain immature DCs, the CD14+ cells were incubated for 5 days in the complete medium (Corning Inc., Corning, NY, USA) supplemented with 100 U/mL of penicillin, 100 μg/mL of streptomycin (Thermo Fisher Scientific Inc.), and 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) containing 50 ng/mL of granulocyte-monocyte colony-stimulating factor (R&D Systems, Minneapolis, MN, USA), 20 ng/mL of interleukin 4 (IL-4) (R&D Systems), and 20 ng/mL of transforming growth factor beta (TGF-β) (Invitrogen, Grand Island, NY, USA). The morphology of the DCs was examined by optical microscopy. On 5 days of DC culture, the immature MoDCs were washed extensively with Dulbecco phosphate buffered saline (PBS; Invitrogen), and pulsed with Pep19 (10 μg/mL) in a tolerogenic milieu for 5 hours via the addition of a cytokine cocktail consisting of IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), TGF-β, and retinoic acid (1 μM, Sigma-Aldrich).
7
Isolation and Differentiation of MDMs
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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation using Percoll gradients and resuspended in RPMI (GE Healthcare, Parramatta, NSW, Australia) supplemented with 10% (v/v) foetal bovine serum (FBS; Bovogen Biologicals, Keilor East, VIC, Australia), 1% (v/v) streptomycin (Thermo Fisher Scientific, North Ryde, NSW, Australia), 2 mM L-glutamine (Thermo Fisher Scientific), 20 mM HEPES (GE Healthcare) at 4 × 106 cells/mL. PBMCs were dispensed in a 24-well plate at 2 × 106 cells/well and incubated for 2 h at 37 °C to allow the monocytes to adhere. The wells were washed three times with pre-warmed phosphate buffered saline (PBS) and the monocytes differentiated into MDMs over 12 days in culture media supplemented with 2 ng/mL granulocyte macrophage-colony stimulating factor (GM-CSF) (Sigma-Aldrich, Castle Hill, NSW, Australia) as described previously [17 (link)]. The media was replaced every 3 to 4 days. MDM culture supernatant was collected prior to the efferocytosis assay and stored at − 80 °C.
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