Cells were clone sorted into 96-well round-bottom plates. HSCs were cultured in S-Clone (Iwai North America Inc.) supplemented with 0.75% AlbuMAX-I (Gibco), 1x penicillin/streptomycin, 50 mM 2-mercaptoethanol (Invitrogen) and the following cytokines: 20 ng/ml mouse stem cell factor, 20 ng/ml mouse thrombopoietin, 20 ng/ml mouse IL-12. Myeloid progenitors and c-kit enriched cells were cultured in Dulbecco’s modified Eagle’s medium and F-12 medium (Gibco and Invitrogen) supplemented with 10% fetal calf serum (Hyclone and Thermo Scientific), 1x penicillin/streptomycin, 2 mM GlutaMAX, 50 mM 2-mercaptoethanol (Invitrogen), and the following cytokines: 20 ng/ml mouse stem cell factor, 20 ng/ml mouse thrombopoietin, 20 ng/ml mouse IL-3, 20 ng/ml mouse granulocyte macrophage colony-stimulating factor (all purchased from PeproTech). c-kit enriched bone marrow (2×106 cells/ml) were exposed for 4 hours to 10 μM ATMi (KU55933, Selleckchem). All cells were kept in a 5% O2 incubator.
Mouse granulocyte macrophage colony stimulating factor
Manufactured by Thermo Fisher Scientific
Sourced in United States
Mouse granulocyte macrophage colony-stimulating factor is a recombinant protein that stimulates the production and function of granulocytes and macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Albumax 1, by Thermo Fisher Scientific (2 mentions)
Mouse stem cell factor, by Thermo Fisher Scientific (2 mentions)
Atmi ku 55933, by Selleck Chemicals (2 mentions)
Mouse thrombopoietin, by Thermo Fisher Scientific (2 mentions)
Mouse il 12, by Thermo Fisher Scientific (2 mentions)
Mouse il 3, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
C57bl 6, by Orient Bio (1 mentions)
Rbc lysing buffer, by Merck Group (1 mentions)
C57bl 6 mice, by Orient Bio (1 mentions)
Rpmi 1640 medium, by Nacalai Tesque (1 mentions)
Mouse il 4, by Thermo Fisher Scientific (1 mentions)
Cxcr4f f, by Jackson ImmunoResearch (1 mentions)
Cd11c cre, by Jackson ImmunoResearch (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Nacalai Tesque (1 mentions)
8 protocols using mouse granulocyte macrophage colony stimulating factor
1
Hematopoietic Stem Cell Culture Protocol
2
Dendritic Cell Culture Protocol
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C57BL/6 mice were purchased from OrientBio (Gyeonggi-do, Korea) and TLR2- or MyD88-deficient mice with C57BL/6 background were kindly provided by Prof. Shizuo Akira (Osaka University, Osaka, Japan). DCs were prepared as previously described (30 (link)). Briefly, bone marrow cells were obtained by flushing the marrow space of tibiae and femurs with phosphate-buffered saline (PBS). After removing red blood cells (RBCs) using an RBC-lysing buffer (Sigma-Aldrich), bone marrow cells were cultured with RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin in the presence of 20 ng/ml of mouse granulocyte/macrophage–colony stimulating factor (Peprotech, Rocky Hill, NJ, USA) and 50 µM of 2-mercaptoethanol (Sigma-Aldrich) for 6 days at 37°C. On day 6 post-culture, DCs were collected by centrifugation and used for experiments.
3
Generation and Characterization of Murine Dendritic Cells
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C57BL/6N mice were obtained from Nihon SLC (Hamamatsu, Japan). As previously described (Tanegashima et al., 2010 ), Cxcl14−/− male and Cxcl14+/− female mice with a C57BL/6 N background were crossed to produce Cxcl14+/− and Cxcl14−/− mice. LysM-Cre knock-in mice (Clausen et al., 1999 (link)) were obtained from RIKEN (RBRC02302), and CD11c-Cre (stock number 007567) and Cxcr4F/F (stock number 008767) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Tlr9-KO mice (Hemmi et al., 2000 (link)) were obtained from Oriental Bioservice, Inc. (Kyoto, Japan).
BMDCs were prepared from 6-week-old C57BL/6N, Tlr9-KO, and Cxcr4-CKO mice by culturing bone marrow cells in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), mouse granulocyte-macrophage colony stimulating factor (10 ng/ml; Peprotech, Rocky Hill, NJ), and mouse IL-4 (10 ng/ml; Peprotech) for 10 days. Splenocytes were prepared from spleen removed from 6 to 8 week-old C57BL/6N mice. Red blood cells were lysed in RBC lysis buffer (140 mM NH4Cl, 17 mM Tris-HCl, pH 7.6). All mice were housed in a pathogen-free animal facility under a 12 h light/dark cycle. All experimental procedures were pre-approved by the ethical committee of Tokyo Metropolitan Institute of Medical Science.
BMDCs were prepared from 6-week-old C57BL/6N, Tlr9-KO, and Cxcr4-CKO mice by culturing bone marrow cells in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), mouse granulocyte-macrophage colony stimulating factor (10 ng/ml; Peprotech, Rocky Hill, NJ), and mouse IL-4 (10 ng/ml; Peprotech) for 10 days. Splenocytes were prepared from spleen removed from 6 to 8 week-old C57BL/6N mice. Red blood cells were lysed in RBC lysis buffer (140 mM NH4Cl, 17 mM Tris-HCl, pH 7.6). All mice were housed in a pathogen-free animal facility under a 12 h light/dark cycle. All experimental procedures were pre-approved by the ethical committee of Tokyo Metropolitan Institute of Medical Science.
4
Hematopoietic Stem Cell Culture Protocol
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Cells were clone sorted into 96-well round-bottom plates. HSCs were cultured in S-Clone (Iwai North America Inc.) supplemented with 0.75% AlbuMAX-I (Gibco), 1x penicillin/streptomycin, 50 mM 2-mercaptoethanol (Invitrogen) and the following cytokines: 20 ng/ml mouse stem cell factor, 20 ng/ml mouse thrombopoietin, 20 ng/ml mouse IL-12. Myeloid progenitors and c-kit enriched cells were cultured in Dulbecco’s modified Eagle’s medium and F-12 medium (Gibco and Invitrogen) supplemented with 10% fetal calf serum (Hyclone and Thermo Scientific), 1x penicillin/streptomycin, 2 mM GlutaMAX, 50 mM 2-mercaptoethanol (Invitrogen), and the following cytokines: 20 ng/ml mouse stem cell factor, 20 ng/ml mouse thrombopoietin, 20 ng/ml mouse IL-3, 20 ng/ml mouse granulocyte macrophage colony-stimulating factor (all purchased from PeproTech). c-kit enriched bone marrow (2×106 cells/ml) were exposed for 4 hours to 10 μM ATMi (KU55933, Selleckchem). All cells were kept in a 5% O2 incubator.
5
Isolation and Culture of Murine and Human Immune Cells
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Murine peritoneal macrophages were prepared by intraperitoneal injection of thioglycollate. The peritoneal cavity was infused with 5–10 ml PBS at 5 days after injection. The fluid-filled cavity was gently shaken and fluid was withdrawn19 (link). Bone marrow-derived DCs (BMDCs) were prepared from bone marrow suspensions from the femurs and tibias in mice as described56 (link). Bone marrow cells were cultured in 10 ng ml−1 mouse granulocyte–macrophage colony-stimulating factor (PeproTech) and the 10-day-cultured BMDCs were used for the experiment. THP-1 cells (RIKEN) were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 2-ME (Invitrogen), L -glutamine and penicillin/streptomycin (Nacalai). HEK293 cells (RIKEN) were maintained in DMEM supplemented with 10% FBS, L -glutamine and penicillin/streptomycin. To prepare human blood monocytes, human peripheral blood mononuclear cells were isolated from heparinized whole blood using Ficoll-Paque PLUS (GE Healthcare) and then adhered on plastic flasks (Iwaki) for 3 h.
6
Treg Proliferation Assay with CFSE
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For Treg proliferation assays, FACS-sorted Foxp3.GFP+CD4+ Tregs were labeled with CFSE. CFSE-labeled Tregs (1×104 cells/well) and MACS-sorted dendritic cells (DC)s (1×105 cells/well) were plated in 200 μl T cell media (MEM-α with 10% FBS, 1% penicillin/streptomycin, 10 mM HEPES, and 1×10−5 M 2-mercaptoethanol) with mouse granulocyte macrophage colony stimulating factor (10 ng/ml; PeproTech, Rocky Hill, NJ) and human IL-2 (50 U/ml; PeproTech) in 96-well flat bottom plates. CD11c+ DCs were obtained from spleens of mice subcutaneously injected 8–10 days prior with FLT3L-expressing EL4 cells. Cells were cultured with or without the indicated factors at 37°C and analyzed by flow cytometry 4 days later.
7
Isolation and Differentiation of Murine BMDMs
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The murine BMDMs of WT and Trem2 -/-mice were isolated from the femur and tibia in single cell suspension and seeded in 6-well plates (2.5 × 10 6 cells per well) with Dulbecco's modi ed eagle medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, USA), 1% penicillin/streptomycin and 20 ng/ml mouse granulocyte macrophage-colony stimulating factor (PeproTech, USA). These cells were differentiated into macrophage for another 6~7 days with replacement of fresh growth media in every 3 days. Cells were maintained in a humidi ed incubator (Thermo Fisher Scienti c, USA) with 5% CO 2 at 37 °C. Then BMDMs were harvest or stimulated for further experiments.
8
Isolation and Differentiation of Murine Macrophages
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Both ends of mouse femurs and tibias were cut and flushed with 5 mL of RPMI 1640 (Invitrogen, Carlsbad, CA) by using a 27-gauge  1 / 2 -inch needle (Terumo, Tokyo, Japan). Bone marrow cells were strained (40-mm mesh) (BD Biosciences, San Jose, CA) and seeded in complete medium that consisted of RPMI 1640 that contained 10% fetal bovine serum, 1 nonessential amino acids (Gibco), 20 mmol/L HEPES (Gibco), and 10 ng/mL mouse granulocyte-macrophage colony-stimulating factor (PeproTech, Rocky Hill, NJ) for macrophage differentiation. One-half of culture medium was replaced every 2 days with fresh complete medium. Differentiated macrophages were harvested after 7 to 10 days with Cellstripper (Corning Cellgro, Manassas, VA) and used for virus infection experiments.
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