BMDCs were obtained by washing the tibiae and femurs of mice with cold PBS and separating cells using a 70-μm cell strainer. The cells were seeded in a 24-well plate (106 cells/well) with conditioned medium (RPMI with 1% penicillin/streptomycin, 10% FBS, glutamine, 1% HEPES, 1% sodium pyruvate, 1% non-essential amino acids, and 50μM β-mercaptoethanol) supplemented with GM-CSF (20 ng/mL, R&D Systems), or GM-CSF VLPs (LD + GM-CSF + 4-1BBL or LD + GM-CSF + OX40L, 5 × 109 particle/mL), and IL-4 (20 ng/mL, R&D Systems). The cells were characterized by flow cytometry labeling surface markers CD11c (FITC anti-mouse CD11c, BioLegend), MHC class II (PE anti-mouse MHC II, BioLegend) and CD86 (APC anti-mouse CD86, BioLegend). The data were analyzed using the ACEA NovoCyte 2000 flow cytometer.
Gm csf
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Germany, France, China, Israel, Canada, Sweden
GM-CSF is a cytokine that stimulates the production and differentiation of granulocytes and macrophages from bone marrow progenitor cells. It is a key regulator of granulopoiesis and macrophage function.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by R&D Systems (178 mentions)
M csf, by R&D Systems (56 mentions)
Interleukin 6 (il 6), by R&D Systems (49 mentions)
Tnf α, by R&D Systems (41 mentions)
Lipopolysaccharides (lps), by Merck Group (36 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (31 mentions)
Il 1β, by R&D Systems (28 mentions)
Rpmi 1640, by Thermo Fisher Scientific (22 mentions)
Ifn γ, by R&D Systems (20 mentions)
Interleukin 3 (il 3), by R&D Systems (20 mentions)
G csf, by R&D Systems (20 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (20 mentions)
Streptomycin, by Thermo Fisher Scientific (18 mentions)
Penicillin, by Thermo Fisher Scientific (18 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (17 mentions)
Cd14 microbead, by Miltenyi Biotec (16 mentions)
L glutamine, by Thermo Fisher Scientific (14 mentions)
Stem cell factor (scf), by R&D Systems (14 mentions)
Gm csf, by Thermo Fisher Scientific (13 mentions)
Il 10, by R&D Systems (11 mentions)
599 protocols using gm csf
1
Mouse Bone Marrow-Derived Dendritic Cell Culture
2
In Vitro and In Vivo Differentiation of BMPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the differentiation patterns of BMPs in vitro, sorted total BMPs, cMoPs, MDPs, CD135+ BMPs, and CD115−CD135− BMPs (1 × 104 cells/well) were cultured for 4 to 6 days under specific conditions as follows: GM-CSF, GM-CSF plus IL-4, or GM-CSF plus IFN-γ (all from R&D systems). All recombinant cytokines were used at 20 ng/ml, and the culture medium was refreshed every 2 days.
To analyze the differentiation patterns of cMoPs in vivo, sorted cMoPs and non-cMoPs (2 × 105 cells each/mouse) were adoptively transferred into LCMV-Arm-infected recipient mice at day 5 p.i., Donor cells were analyzed at day 3 post transfer (day 8 p.i.).
To analyze the differentiation patterns of cMoPs in vivo, sorted cMoPs and non-cMoPs (2 × 105 cells each/mouse) were adoptively transferred into LCMV-Arm-infected recipient mice at day 5 p.i., Donor cells were analyzed at day 3 post transfer (day 8 p.i.).
3
Generation of Bone Marrow-Derived Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow derived DCs were generated as we have reported [57 (link),58 (link)]. Briefly, bone marrow cells were collected from femurs and tibias of transgenic NOD.CD11cStat5b-CA and NOD mice. One million cells were plated in 10 mL bacterial Petri dishes (UltiDent Scientific, St. Laurent, QC, Canada) and cultured in RPMI 1640 medium supplemented with 10% heat inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and β-mercaptoethanol (50 μM) in the presence of GM-CSF (5 ng/mL) and IL-4 (4.5 ng/mL) (R&D Systems Inc. Minneapolis, MN, USA). At day 3, fresh medium (10 mL) supplemented with GM-CSF (5 ng/mL) and IL-4 (4.5 ng/mL) was added to the culture. At day 5, half of the medium was removed and replaced with fresh medium supplemented with GM-CSF (5 ng/mL) and IL-4 (4.5 ng/mL). At day 7, non-adherent cells were gently harvested, pooled, and left unstimulated or exposed to GM-CSF (50 ng/mL) (R&D Systems Inc. Minneapolis, MN, USA) for 48 h. Over 92% of non-adherent cells were CD11c+. All experiments were performed with relevant guidelines and regulations. All mice were housed under pathogen-free conditions at the Faculty of Medicine in accordance with the guidelines of the Institutional Animal Care Committee of the University of Sherbrooke (Protocol # 93-18), Sherbrooke, QC, Canada.
4
Isolation and characterization of murine and human MDSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow mononuclear cells cells (BMMCs) were ushed out the bone marrow cavity of tibias and femurs with α-minimum essential medium (Invitrogen). BMMCs were cultured in 6 well plate at 1 × 10 6 cells /mL in complete medium supplemented with GM-CSF (20 ng/mL) and M-CSF (20 ng/mL) (both from R&D Systems). After three days, they were harvested and stained with CD11c, CD11b, and Gr-1 antibodies after blocking Fc receptors (all from BD Biosciences). CD11c-, CD11b+, and Gr-1 + MDSC populations were sorted using a MoFlo cell sorter (Beckman Coulter). Human PBMC were cultured in 6 well plate at 1 × 10 6 cells/ml in complete medium supplemented with GM-CSF(20 ng/ml) and IL-6 (20 ng/ml) (both from R&D Systems). After three days, Lineage-/HLA-DR-/CD33+/CD11b + human MDSC subsets were sorted using a MoFlo cell sorter (Beckman Coulter). The purity of the sorted MDSCs was > 95%.
5
Culture and Activation of AML Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KG-1 (CCL-246, KG-1 AML, Lot 63011477) and MUTZ-3 (ACC 295) cell lines were purchased from ATCC (Rockville, USA) and DSMZ (Braunschweig, Germany), respectively. Both cell lines were cultured in a humidified incubator at 37 °C with 5% CO2 as described35 (link)–41 (link). Briefly, KG-1 cells were cultured in RPMI 1640 Medium (RPMI; Gibco, Thermo Fisher Scientific, MA, USA) supplemented with 20% (v/v) Fetal Bovine Serum (FBS; Hyclone, Logan, USA) and penicillin/streptomycin (100 U/mL); MUTZ-3 cells were cultured with α-Minimum Essential Medium (α-MEM; Gibco, Thermo Fisher Scientific) supplemented with 20% (v/v) FBS, 100 U/mL penicillin/streptomycin and 25 IU/mL GM-CSF (R&D systems, Minneapolis, USA). MUTZ-3 maturation was stimulated as previously described34 (link),38 (link),41 (link). Briefly, 2–4 × 105 cells/mL were seeded in complete media with 50 ng/mL GM-CSF and 20 ng/mL Interleukin-4 (IL-4, R&D systems) for 7 days. For mature MUTZ-3 activation 12 ng/mL TNF (R&D Systems) or 1 μg/ml of LPS (Sigma-Aldrich) were added independently at the end of day 5 and then cells were harvested at day 7 and stored at − 80 °C.
6
Differentiation of Mouse and Human Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For mouse BMDC induction, bone marrow cells from C57BL/6J mice were cultured in RPMI 1640 culture medium containing 10% FBS, 1% Antibiotic-Antimycotic, 10 mM Hepes, 50 μM β-mercaptoethanol, 2 mM l -glutamine (all from Gibco), granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/ml), and IL-4 (10 ng/ml; PeproTech). The culture medium was partially replaced every 2 days, and the suspended cells were harvested on day 6. For human moDCs induction, the PBMCs were prepared using Ficoll gradient centrifugation (STEMCELL Technologies) and cultured for 6 days in RPMI 1640 culture medium supplemented with GM-CSF (100 ng/ml) and IL-4 (30 ng/ml; R&D Systems). Fresh culture medium was replaced every 3 days, and the suspended cells were harvested on day 6. For the induction of THP-1–derived DCs, THP-1 cells were cultured for 6 days in RPMI 1640 culture medium supplemented with GM-CSF (100 ng/ml) and IL-4 (30 ng/ml; R&D Systems), fresh culture medium was added every 2 days, and the suspended cells were harvested on day 6.
7
Isolation and Characterization of MDSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow mononuclear cells cells (BMMCs) were ushed out the bone marrow cavity of tibias and femurs with α-minimum essential medium (Invitrogen). BMMCs were cultured in 6 well plate at 1 × 10 6 cells /mL in complete medium supplemented with GM-CSF (20 ng/mL) and M-CSF (20 ng/mL) (both from R&D Systems). After three days, they were harvested and stained with CD11c, CD11b, and Gr-1 antibodies after blocking Fc receptors (all from BD Biosciences). CD11c-, CD11b+, and Gr-1+ MDSC populations were sorted using a MoFlo cell sorter (Beckman Coulter). Human PBMC were cultured in 6 well plate at 1 × 10 6 cells/ml in complete medium supplemented with GM-CSF(20 ng/ml) and IL-6 (20 ng/ml) (both from R&D Systems). After three days, Lineage-/HLA-DR-/CD33+/CD11b+ human MDSC subsets were sorted using a MoFlo cell sorter (Beckman Coulter). The purity of the sorted MDSCs was >95%.
8
Quantification of Growth Factors and Cytokines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure the concentration of HGF, TGF-α, GM-CSF, and IL-8 in the culture medium, we used a human HGF, TGF-α, GM-CSF (R&D Systems), and IL-8 ELISA (Elisa Tech, Aurora, CO) according to the manufacturers' instructions. For the Western blotting analysis, polyacrylamide gradient gels (8–16%; Invitrogen, Carlsbad, CA) were run in Tris-glycine buffer to separate the proteins. Protein loading was normalized to GAPDH. The primary antibodies to phosphorylated EGFR (Y1086) and EGFR antibody were from Abcam (Cambridge, MA).
9
Co-culture of Macrophages and Liver Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfection: SMMC-7721, a human liver cancer cell line, was purchased from Thermo Fisher Scientific Inc. (Shanghai, China, 61870036), THP-1, a human monocytic leukemia cell line, was purchased from Wuhan Punoise Life Technologies Co., Ltd. (Wuhan, China, CL-0233), RAW264.7, a mouse monocyte macrophage leukemia cell line, was purchased from Wuhan Punoise Life Technologies Co., Ltd. (Wuhan, China,CL-0190). SHP-1 mimic and NC mimic were purchased from Genecopoeia (Guangzhou, China). Mimics were diluted with serum-free dilution solution and mixed thoroughly with EntransterTM-R4000. After incubating at room temperature for 15 min, the transfection complex was dropped onto THP-1 cells and incubated for 24 h.
Macrophage were seeded at a density of 1 × 106 cells/mL in RPMI 1640 complete medium treated with PMA (100 ng/mL) for 6 h, followed by the addition of IL-13 (20 ng/mL) and IL-4 (20 ng/mL) for 18 h to generate M2 polarized macrophages.
The co-culture system of macrophages and HCC cells: PMA-treated THP-1 macrophages were seeded into the upper insert of a 6-well Transwell device and co-cultured with SMMC-7721 cells in the lower chamber of a 6-well plate. The two cell types did not directly contact each other. After co-culturing for 16 h, the cells were co-cultured with GM-CSF or without GM-CSF (25 ng/mL R&D Systems) and under normoxic (20% O2) or hypoxic (1% O2) conditions for 24 h.
Macrophage were seeded at a density of 1 × 106 cells/mL in RPMI 1640 complete medium treated with PMA (100 ng/mL) for 6 h, followed by the addition of IL-13 (20 ng/mL) and IL-4 (20 ng/mL) for 18 h to generate M2 polarized macrophages.
The co-culture system of macrophages and HCC cells: PMA-treated THP-1 macrophages were seeded into the upper insert of a 6-well Transwell device and co-cultured with SMMC-7721 cells in the lower chamber of a 6-well plate. The two cell types did not directly contact each other. After co-culturing for 16 h, the cells were co-cultured with GM-CSF or without GM-CSF (25 ng/mL R&D Systems) and under normoxic (20% O2) or hypoxic (1% O2) conditions for 24 h.
10
Isolation and Activation of Human and Murine Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human myeloid DCs were isolated from peripheral blood. After Ficoll-Hypaque (PAA, GE, United Kingdom) separation, lymphocytes were sorted using CD14 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD14 cells were cultured in RPMI 1640 containing 10% FBS, 50 ng/mL GM-CSF (R&D, Minnesota, USA), and 10 ng/mL IL-4 (R&D Systems, Minneapolis, MN, USA) overnight. The following day, non-adherent cells were removed by gentle pipetting and adherent cells were cultured in the same medium for two additional days. DCs were activated using lipopolysaccharides (LPS) (Sigma-Aldrich, St. Louis, MO, USA) for 24h.
Murine DCs were isolated from tumor-draining lymph nodes. After removal of red blood cells through lysis, DCs were cultured overnight in RPMI 1640 containing 10% FBS, 1000 U/mL GM-CSF (R&D Systems), and 1000 U/mL IL-4 (R&D Systems). The following day, non-adherent cells were removed by gentle pipetting and adherent cells were cultured in the same medium for two additional days. The cells were sorted using CD11c magnetic beads (Miltenyi Biotec). CD11c+cells were then activated with LPS (Sigma-Aldrich) for 24 h.
Murine DCs were isolated from tumor-draining lymph nodes. After removal of red blood cells through lysis, DCs were cultured overnight in RPMI 1640 containing 10% FBS, 1000 U/mL GM-CSF (R&D Systems), and 1000 U/mL IL-4 (R&D Systems). The following day, non-adherent cells were removed by gentle pipetting and adherent cells were cultured in the same medium for two additional days. The cells were sorted using CD11c magnetic beads (Miltenyi Biotec). CD11c+cells were then activated with LPS (Sigma-Aldrich) for 24 h.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!