Prestimulated human NK cells were coincubated with or without hyphae of A. fumigatus for 4 hours. NK cells at time point 0 hours served as control. Cells were lysed with RIPA Lysis and Extraction Buffer Kit (Thermo Scientific) containing HALT Phosphatase and Protease Inhibitor Cocktails (both Thermo Scientific). Proteins were separated by SDS-PAGE using 4-15% Mini Protean TGX Precast Protein Gels (Bio-Rad). Proteins were blotted onto a nitrocellulose membrane which was blocked with 5% skim milk-PBS solution. Mouse monoclonal antibodies against human IFN-γ (1:200; LifeSpan BioSciences, Seattle, USA), human perforin (1:1000, LifeSpan BioScience), human GM-CSF (1:1000, R&D Systems), and human GAPDH (1:20000; Biolegend) were used as primary antibodies, goat anti-mouse IgG-HRP antibody (1: 100000; Abcam, Cambridge, UK) as secondary antibody. For visualization, Gel Doc™ XR+ System (Bio-Rad) and Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare Life Sciences, Little Chalfont, UK). For comparison of intracellular protein levels, the ratio of the protein of interest and GAPDH was calculated by determination of relative band intensities using Image Lab 5.0 software (Bio-Rad).
Human gm csf
Manufactured by R&D Systems
Sourced in United States
Human GM-CSF is a recombinant protein that functions as a cytokine. It stimulates the production and release of granulocytes and macrophages from bone marrow.
Automatically generated - may contain errors
Lab products found in correlation
Human il 4, by R&D Systems (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Gm csf, by R&D Systems (2 mentions)
Glutamax 1, by Thermo Fisher Scientific (2 mentions)
Mifepristone, by Merck Group (2 mentions)
Methanol, by Avantor (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Facsverse, by BD (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Bio plex, by Bio-Rad (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ripa lysis and extraction buffer kit, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Goat anti mouse igg hrp antibody, by Abcam (1 mentions)
Amersham ecl select western blotting detection reagent, by GE Healthcare (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
Recombinant mouse gm csf, by R&D Systems (1 mentions)
21 protocols using human gm csf
1
Quantification of NK Cell Proteins
2
Isolation and differentiation of human and mouse dendritic cells
3
Human Monocyte-to-Macrophage Differentiation
4
Quantifying GM-CSFRα Expression on Neutrophils
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TF‐1 cells (R&D Systems) that had been maintained in human GM‐CSF (4 ng/ml, R&D Systems) were washed 3 times to remove GM‐CSF. GM‐CSFRα expression was assessed on the cells following culture for a further 18 hr in the presence or absence of GM‐CSF (4 ng/ml).
GM‐CSFRα expression was quantified on human neutrophils cultured in the presence of GM‐CSF (0.001‐10 ng/ml, R&D Systems), LPS (100 ng/ml, Sigma Aldrich UK, Poole, UK), TNFα (20 ng/ml, R&D Systems) or appropriate vehicle control. In certain experiments neutrophils were pretreated for 30 min with CAM‐3001 (blocking antibody specific to human GM‐CSFRα) or for 1 hr with the proteasomal inhibitor MG132 (20 μM, Sigma Aldrich), brefeldin A (10 μg/ml) to block lysosomal degradation, or the transcriptional inhibitor actinomycin D (2 μg/ml); in certain experiments IL‐8 was measured in the supernatants using an in‐house ELISA.20 GM‐CSFRα levels were also quantified on blood neutrophils and BALF neutrophils derived from patients with ARDS.
GM‐CSFRα expression was quantified on human neutrophils cultured in the presence of GM‐CSF (0.001‐10 ng/ml, R&D Systems), LPS (100 ng/ml, Sigma Aldrich UK, Poole, UK), TNFα (20 ng/ml, R&D Systems) or appropriate vehicle control. In certain experiments neutrophils were pretreated for 30 min with CAM‐3001 (blocking antibody specific to human GM‐CSFRα) or for 1 hr with the proteasomal inhibitor MG132 (20 μM, Sigma Aldrich), brefeldin A (10 μg/ml) to block lysosomal degradation, or the transcriptional inhibitor actinomycin D (2 μg/ml); in certain experiments IL‐8 was measured in the supernatants using an in‐house ELISA.
5
Macrophage Differentiation from Monocytes
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Human mononuclear cells were isolated from volunteer donor blood buffy coats (Sanquin), peripheral blood from healthy controls and RA patients, and from synovial fluid of RA patients by gradient centrifugation with Lymphoprep (Axis-Shield PoPAS) and monocytes were further isolated by Percoll gradient separation (GE Healthcare). Monocytes were differentiated into macrophages in IMDM/10 % fetal calf serum (FCS) supplemented with 100 μg/ml gentamycin (Invitrogen), in the presence of human GM-CSF (5 ng/ml, R&D Systems), CSF-1 (25 ng/ml, R&D Systems), or IL-34 (25 ng/ml, provided by Five Prime Therapeutics Inc.) for 7 days.
6
PBMC Isolation and Monocyte Differentiation
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Peripheral blood mononuclear cells (PBMCs) from the patient blood were separated using density gradient centrifugation. Briefly, whole blood was diluted with PBS (1:1) and layered on top of Lymphoprep™ medium in a SepMate™ 50ml tube (Stem Cell Technologies) prior to centrifugation. The buffy coat layer containing the enriched PBMCs was collected, and the cells were incubated with warm red blood cell lysis buffer for 10 minutes before being washed with cold PBS. Isolated PBMCs were cultured in RPMI 1640, supplemented with 10% heat inactivated FCS, 2 mM GlutaMax-1 (Life Technologies), 100 U/ml penicillin, and 100 mg/ml streptomycin and were treated with human GM-CSF (20 ng/ml, R&D Systems) or Dex (100 nM, Sigma-Aldrich), either alone or in combination for 16 h.
7
Molecular Signaling Pathway Analysis
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Reagents included human GM-CSF (R&D Systems, Minneapoilis, MN), human M-CSF (Chiron, Emeryville, CA), dexamethasone (Dex), and mifepristone (Sigma-Aldrich, St. Louis, MO). Antibodies for Western blotting were against phospho-p38 (D3F9), p38 (D13E1), phospho-JNK1/2 (81E11), JNK1/2 (9252), phospho-ERK1/2 (D13.14.4E), ERK1/2 (137F5), caspase-3 (9662), caspase-8 (D35G2), Bcl2 (50E3), Bcl-xL (54H6), Bid (2002), Bak (D4E4), Bax (D2E11), Bim (C34C5), phospho-Bad S112 (40A9), Bad (D24A9), phospho-RSK S380 (D3H11) and RSK (32D7) (Cell Signaling Technologies, Danvers, MA), and β-actin (A5316) (Sigma-Aldrich).
8
Cell Culture Protocols for HIV Research
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HEK293T cells (AIDS Research and Reference Reagents Program, Cat #3522) and TZM-bl cells (AIDS Research and Reference Reagents Program, Cat#8129) were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technology) with 10% fetal bovine serum (FBS) and penicillin-streptomycin. The cell lines U937 and K562 (ATCC) were provided by Dr. Donald Small (Johns Hopkins University) and cultured in RPMI medium with 10% FBS. SupT1 cells were grown in RPMI medium supplemented with 10%FBS. Peripheral blood mononuclear cells (PBMC) were obtained from buffy-coat by Ficoll density gradient centrifugation. Monocytes enriched from PBMC by positive selection using CD14 MicroBeads were differentiated into macrophages by culturing in RPMI supplemented with 10% FBS, 50 ng/ml human GM-CSF (R&D Systems) 100 U/ml penicillin, and 100 μg/ml streptomycin. shRNA-silenced cell lines were generated according to the manufacturer's instructions (Open biosystem) and selected for resistance to puromycin. All cultured cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
9
Phosphoflow Analysis of JMML iPSCs
10
Generation of MAC-CYP Cells from Bone Marrow
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The MAC-CYP cell generation was slightly modified from a previous report [25 (link)]. Briefly, after lysis of the red blood cells, bone marrow nucleated cells were collected and used for purification of CD11b+Gr1+ monocytes using the MACS technique. Gr1+ cells were positively selected using APC-conjugated anti-Gr1 antibody and anti-APC Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The positively selected cells were cultured in RPMI medium 1640 (Invitrogen) containing 10% FBS, human GM-CSF (R&D Systems), human G-CSF (R&D Systems), and human IL-13 (R&D Systems), each at 100 ng/mL for seven days. After seven days’ culture, the cells were transduced with indicated lentiviral vectors at a multiplicity of infection (MOI) of 5. Twenty-four hours later, the virus was removed, and culture media were replenished. The cells were cultured for another 24 h and examined for transduction efficiency under a fluorescence microscope. When necessary, the above transduction procedure was repeated one more time before use.
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