Mgm csf

Manufactured by Thermo Fisher Scientific

Sourced in United States

The MGM-CSF is a specialized lab equipment designed for cell culture applications. It functions as a Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) production system, enabling the growth and expansion of specific cell types. The core purpose of this product is to facilitate the in vitro cultivation of relevant cell lines for research and experimental purposes.

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29 protocols using mgm csf

Myeloid Cell Proliferation Assay

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FDC-P1 cells (murine myeloid cells, CRL-12103, American Type Culture Collection, VA, USA) were exposed to increasing concentrations of mGM-CSF generated in vitro by rSIV.F/HN-mGM-CSF (0.001–10 ng/mL), and values were compared with cells exposed to recombinant mGM-CSF (PeproTech, London, UK). The “One Solution” cell proliferation assay (Promega) was used to estimate cell numbers according to the manufacturer’s recommendation.

Lund-Palau H., Juarez-Molina C.I., Meng C., Bhargava A., Pilou A., Aziz K., Clarke N., Atsumi N., Ashek A., Wilson M.R., Takata M., Padley S., Gill D.R., Hyde S.C., Morgan C., Alton E.W, & Griesenbach U. (2022). Correction of a chronic pulmonary disease through lentiviral vector-mediated protein expression. Molecular Therapy. Methods & Clinical Development, 25, 382-391.

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Differentiation of Murine Bone Marrow Dendritic Cells

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The bone marrow cells obtained from the femur of naïve C57BL/6 mice were transferred to a 100 mm petri dish and cultured in an RPMI medium supplemented with 200 U/mL of mGM-CSF (Peprotech, Rocky Hill, NJ, USA). Six days later, the cells were analyzed for the expression of CD11b, CD11c, and MHC II by flow cytometry before further experiments.

Lim Y., Kim S., Kim S., Kim D.I., Kang K.W., Hong S.H., Lee S.M., Koh H.R, & Seo Y.J. (2020). n-3 Polyunsaturated Fatty Acids Impede the TCR Mobility and the TCR–pMHC Interaction of Anti-Viral CD8+ T Cells. Viruses, 12(6), 639.

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Murine Dendritic Cell Culture Protocol

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A20 B cell lymphoma cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained via ATCC protocol.
Dendritic cells preparation was performed as previously described and modified [4 (link)]. Bone marrow–derived murine dendritic cells were generated by culturing bone marrow cells from the femur and tibiae of BALB/c mice at a starting concentration of 1×10⁶ cells/mL in RPMI-1640/10% fetal bovine serum (FBS; Invitrogen, Carlsbad, California) supplemented with 30 ng/mL of recombinant murine granulocyte macrophage colony-stimulating factor (mGM-CSF; PeproTech, Rocky Hill, NJ) and 3 ng/mL interleukin 4 (mIL-4; PeproTech, Rocky Hill, NJ). Fresh medium supplemented with GM-CSF plus IL-4 was added on day 3, and all of the loosely adherent cells were transferred to Petri dishes on day 6. 2 days later, nonadherent cells and loosely adherent dendritic cells were harvested, washed with PBS.

Zhu X.J., Yang Z.F., Zhou J.Y., Liu L., Sun X.M., Fan Z.F., Hu S.Y., Chen Y.C., Li W.X., Cao M, & Wang L.X. (2015). Progression of Large Lymphoma Is Significantly Impeded with a Combination of Gemcitabine Chemotherapy and Dendritic Cells Intra-Tumor Vaccination. PLoS ONE, 10(7), e0132799.

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Bone Marrow-Derived Dendritic Cell Culture

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Bone marrow-derived dendritic cells were cultured as previously described [19 (link)]. Briefly, bone marrow from the femurs and tibia were collected aseptically from C57BL/6 mice. Cells were cultured for 6 days in RPMI-1640 containing 10% heat-inactivated FBS, 20 ng/ml mGM-CSF (Peprotech), 50 μM β-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were incubated at 5% CO₂ and 37°C. After 3 days, mGM-CSF containing media was supplemented. Loosely adherent cells were collected on the sixth day and used for the further experiment [57 (link)]. Percentage purity of DCs was 65% to 80%.
Total splenocytes were isolated from the spleen of C57BL/6-Tg (TcraTcrb) 1100Mjb/J mice by mechanical disruption. Erythrocytes were lysed by RBC lysis buffer (Sigma) and cells were maintained in RPMI-1640 containing 10% heat-inactivated FBS. Finally, non-adherent cells were collected and were used for mixed lymphocyte proliferation assay.
Anti SIRT2 antibody (Boster, #PA2283), anti NFκB p65 antibody (Cell Signalling, #3034), anti NOS2 antibody (Bimol, #Cay160862), anti Iκα antibody (Santa Cruz Biotech, #A2208) was used for immunoblot and immunofluorescence analysis.

Gogoi M., Chandra K., Sarikhani M., Ramani R., Sundaresan N.R, & Chakravortty D. (2018). Salmonella escapes adaptive immune response via SIRT2 mediated modulation of innate immune response in dendritic cells. PLoS Pathogens, 14(11), e1007437.

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Differentiation of 800-cell EBs

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800-cell EBs were formed in hanging drops in culture medium without LIF. At day 2, EBs were transferred into tissue-culture plates, and the following cytokines were added to induce differentiation: 50 ng/ml m-BMP4, 30 ng/ml m-VEGF164 (both from R&D), 50 ng/ml m-SCF, 50 ng/ml h-Flt3L, 10 ng/ml m-IL-3, 10 ng/ml m-IL-6, and 10 ng/ml m-GM-CSF (all from PeproTech). At day 4 and 8, fresh medium was added with the same cytokine cocktail with the exception of m-BMP4. FACS analyses were performed from day 8 to 12 of culture in most experiments.

Neri T., Muggeo S., Paulis M., Caldana M.E., Crisafulli L., Strina D., Focarelli M.L., Faggioli F., Recordati C., Scaramuzza S., Scanziani E., Mantero S., Buracchi C., Sobacchi C., Lombardo A., Naldini L., Vezzoni P., Villa A, & Ficara F. (2015). Targeted Gene Correction in Osteopetrotic-Induced Pluripotent Stem Cells for the Generation of Functional Osteoclasts. Stem Cell Reports, 5(4), 558-568.

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Culture and Expansion of Genotyped AML Cells

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Thawed cryo-preserved cells from primary AML were cultured in Lonza X-Vivo^TM 15 cell medium (#BE02-060Q Thermo Fisher Scientific) supplemented with Bovine Serum Albumin in Iscove’s MDM (10%; #09300 Stemcell^TM Technologies), Penicillin-Streptomycin (1%; #15140122 Gibco), β-mercaptoethanol (0.1 mM; #31350010 Gibco), L-glutamine (2 mM; #25030149 Gibco), and cytokines h-IL-6 (50 ng/ml; #130-093-032 Miltenyi Biotec), m-SCF (50 ng/µl; #250-03 Peprotech), m-IL-3 (10 ng/ml; #213-13 Peprotech), and m-GM-CSF (10 ng/ml; #315-03 Peprotech). Two clones of each genotype (Cebpa^Δ/p30Tet2^+/+ and Cebpa^Δ/p30Tet2^Δ/Δ) continued to expand beyond 40 days and withstood freeze-thawing, and these clones have been used for further experiments.

Heyes E., Wilhelmson A.S., Wenzel A., Manhart G., Eder T., Schuster M.B., Rzepa E., Pundhir S., D’Altri T., Frank A.K., Gentil C., Woessmann J., Schoof E.M., Meggendorfer M., Schwaller J., Haferlach T., Grebien F, & Porse B.T. (2023). TET2 lesions enhance the aggressiveness of CEBPA-mutant acute myeloid leukemia by rebalancing GATA2 expression. Nature Communications, 14, 6185.

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Generation of Murine Bone Marrow-Derived Dendritic Cells

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Bone marrow cells were collected from the tibias and femurs of C57BL/6 mice and cultured with complete RPMI-1640 medium containing 20 ng/ml mGM-CSF (PeproTech). Fresh medium supplemented with mGM-CSF was added to the culture 3 days later. Immature BMDCs were collected and ready to use on day 7. Naive T cells were isolated from the spleen with a negative CD3⁺ T-cell isolation kit (STEMCELL Technologies).

Liu Y., Cai J., Liu W., Lin Y., Guo L., Liu X., Qin Z., Xu C., Zhang Y., Su X., Deng K., Yan G, & Liang J. (2020). Intravenous injection of the oncolytic virus M1 awakens antitumor T cells and overcomes resistance to checkpoint blockade. Cell Death & Disease, 11(12), 1062.

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Isolation of Bone Marrow-Derived Macrophages

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Bone marrow-derived macrophages were obtained from 4 to 6 week-old Rsad2^+/+ and Rsad2^−/− female mice. The cells were cultured using DMEM complete medium containing 100 ng/ml mGM-CSF (Pepro Tech, USA) and 10% FBS (Corning, NY, USA) in 5% CO₂ incubator at 37 ℃. On day three and five, half of the above complete medium is refreshed. After seven days of culture, we digested the cells with pancreatic enzyme and cultured them in 5% CO₂ cell culture incubator at 37 ℃ for another 24 h before they could be performed.

Liang Y., Liang Y., Wang Q., Li Q., Huang Y., Li R., Pan X., Lie L., Xu H., Han Z., Liu H., Wen Q., Zhou C., Ma L, & Zhou X. (2024). Viperin inhibits interferon-γ production to promote Mycobacterium tuberculosis survival by disrupting TBK1-IKKε-IRF3-axis and JAK-STAT signaling. Inflammation Research, 73(6), 897-913.

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Assessing Myeloid Cell Cytotoxicity and Inflammation

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Bone marrow-derived myeloid cells were obtained from culture of bone marrow cells from adult C57BL/6 mice in BMM media (RPMI with 10% FBS, 50 U/mL Penicillin/Streptomycin 10mM HEPES, 1mM Sodium Pyruvate, 50 μM β-mercaptoethanol and 20 ng/mL mGM-CSF (PeproTech)). Cells were grown in 100 mm x 20 mm tissue-culture dishes. At D3, D6 and D8 the media was renewed. After 9 days, non-adherent cells were collected, washed, counted and re-plated at a density of 250,000 per well in 96-well plates. They were then incubated at 37 degrees with sterilely-filtered bacterial supernatants, which were diluted in BMM media to 30% of the total volume. Cells incubated with the same percentage of corresponding bacterial media (TSB for Staphylcoccus spp, BHI for Corynebacterium spp) in BMM media served as controls. After 2 hours of incubation, media supernatant was collected from each well. Lactate dehydrogenase (LDH) was measured with the Pierce LDH cytotoxicity assay kit (Thermo Scientific) according to manufacturer’s instructions to assess cell death. Values are reported as ΔLDH, meaning the LDH activity of the condition subtracted by the LDH activity of corresponding media control. The concentration of IL-1β was measured in cell culture supernatants using enzyme-linked immunosorbent assay (ELISA) (R&D Systems) according to the manufacturer’s instructions.

Leech J.M., Dhariwala M.O., Lowe M.M., Chu K., Merana G.R., Cornuot C., Weckel A., Ma J.M., Leitner E.G., Gonzalez J.R., Vasquez K.S., Diep B.A, & Scharschmidt T.C. (2019). Toxin-triggered interleukin-1 receptor signaling enables early life discrimination of pathogenic versus commensal skin bacteria. Cell host & microbe, 26(6), 795-809.e5.

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Generating Murine Dendritic Cells

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Mouse bone marrow cells were isolated from 8‐week‐old C57BL/6J male mice (Beijing HFK Bioscience, Beijing, China) and cultured for 7 days in RPMI‐1640 medium (Gibco) containing 10% FBS (Biological Industries), supplemented with 20 ng·mL^–1 murine granulocyte macrophage‐colony stimulating factor (mGM‐CSF; PeproTech), and 20 ng·mL^–1 mIL‐4 (PeproTech) at 37 °C in a 5% CO₂ incubator. The generated DCs were immature and their purity was determined by flow cytometry analysis of CD11c⁺ cells. Animal experiments were approved by the Animal Ethical and Welfare Committee of Shandong University (AEWC number: 18021) and were compliant with the Guide for the Care and Use of Laboratory Animals. Mice were housed in a rectangular mouse cage (area: 635 cm², height: 18 cm) and were kept in a specific pathogen‐free environment under standard experimental conditions (light–dark cycle: 12 h, temperature: 20–22 °C, humidity: 50–70%) with ad libitum access to food and water. Five mice were housed in one cage and were cared for every day.

Li Y., Song Z., Han Q., Zhao H., Pan Z., Lei Z, & Zhang J. (2022). Targeted inhibition of STAT3 induces immunogenic cell death of hepatocellular carcinoma cells via glycolysis. Molecular Oncology, 16(15), 2861-2880.

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