Mouse m csf

Manufactured by Thermo Fisher Scientific

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Mouse M-CSF is a recombinant protein that functions as a growth factor for macrophages. It promotes the differentiation and proliferation of macrophages, which play a crucial role in the immune system. This product is intended for use in research applications.

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M-CSF (1336 citations) Recombinant mouse M-CSF (47 citations) Recombinant mouse macrophage colony stimulating factor (M-CSF; (7 citations) Recombinant soluble mouse M-CSF (3 citations) Mouse M-CSF Recombinant Protein (3 citations) Recombinant mouse RANKL and M-CSF (2 citations) Recombinant soluble mouse M-CSF (Catalog#315-02) (2 citations)

24 protocols using mouse m csf

Macrophage differentiation and stimulation

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Raw264.7 cells were cultured in RMPI-1640 containing 20% (vol/vol) FBS (GIBCO, Invitrogen Inc, Carlsbad, CA, USA) and 1% (vol/vol) antibiotics (100 U/ml penicillin) at 37 °C in 5% CO₂. Raw264.7 stimulated with IL-4 (25 ng/ml; catalog no. 214-14; PeproTech, Rocky Hill, NJ, USA), or with IFN-γ (25 ng/ml; catalog no. 315-05; PeproTech, Rocky Hill, NJ, USA) and LPS (100 ng/ml; catalog no. L2630; Sigma, St Louis, MO, USA) for 24 h. Adherent cells were washed and harvested with trypsin/EDTA (Lonza).
Bone marrow-derived macrophages (BMMs) were isolated, as previously described [60 (link)]. BMMs obtained from Lyz2-Cre + Twist1fl/fl and Lyz2-Cre-Twist1fl/fl mice were cultured in RMPI-1640 containing 10% (vol/vol) FBS, 25 ng/ml mouse M-CSF (catalog no. 315-02; PeproTech, Rocky Hill, NJ, USA), and 1% (vol/vol) penicillin/streptomycin antibiotics for 5 days, Briefly, on day 5, cells were replated in triplicate (3 × 105 cells/well). BMMs were cultured with serum-free medium and treated with IL-4 (25 ng/ml; catalog no. 214-14; PeproTech, Rocky Hill, NJ, USA), or with IFN-γ (25 ng/ml; catalog no. 315-05; PeproTech, Rocky Hill, NJ, USA) and LPS (100 ng/ml; catalog no. L2630; Sigma, St Louis, MO, USA) for 24 h. Adherent cells were washed and harvested with trypsin/EDTA (Lonza).

Wu Q., Sun S., Wei L., Liu M., Liu H., Liu T., Zhou Y., Jia Q., Wang D., Yang Z., Duan M., Yang X., Gao P, & Ning X. (2022). Twist1 regulates macrophage plasticity to promote renal fibrosis through galectin-3. Cellular and Molecular Life Sciences, 79(3), 137.

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Quantifying Intracellular Staphylococcus aureus

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Peritoneal exudate cells (PECs) were collected from mice infected intraperitoneally with 10⁷ CFU of heat-killed S. aureus 48 h and 24 h (Reyes-Robles et al., 2013 (link)). S. aureus MOI 10 were opsonized for 30 min with 50% normal mouse serum (NMS, Innovative Research) in RPMI 1640 medium (Gibco), washed and suspended in medium. PECs were infected with opsonized S. aureus MOI 1 in coated plates for 1 h and 24 h. PECs were lysed with 0.1% saponin and recovered bacteria (CFU) enumerated by serial dilutions.
Gentamicin protection assays were performed in HaCaT or BMDMs isolated from mice and differentiated in the presence of 60 ng/ml of mouse M-CSF (Peprotech). After stimulation, HaCaT 2 hours, BMDMs for 20 minutes with S. aureus MOI 100, cells were washed and treated with gentamicin (500 μg/mL) for 1 h or 24 h. Cells were washed in PBS, lysed with 0.1% saponin and bacteria enumerated by serial dilution on agar plates.

Kitur K., Wachtel S., Brown A., Wickersham M., Paulino F., Peñaloza H.F., Soong G., Bueno S., Parker D, & Prince A. (2016). Necroptosis promotes Staphylococcus aureus clearance by inhibiting excessive inflammatory signaling. Cell reports, 16(8), 2219-2230.

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Murine Bone Marrow-Derived Macrophage Culture

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Bone marrow-derived macrophages were flushed from the tibia and femurs of 8-week-old mice and cultured in α-MEM (KGM119900-1, KeyGEN BioTECH, China) supplemented with 10% FBS, 1% penicillin/streptomycin (Sigma) and 20 ng/mL mouse M-CSF (AF-315-02, Peprotech, USA) for 7 days.

Liu J., Shen D., Wei C., Wu W., Luo Z., Hu L., Xiao Z., Hu T., Sun Q., Wang X., Ding Y., Liu M., Pang M., Gai K., Ma Y., Tian Y., Yu Y., Wang P., Guan Y., Xu M., Yang F, & Li Q. (2022). Inhibition of the LRRC8A channel promotes microglia/macrophage phagocytosis and improves outcomes after intracerebral hemorrhagic stroke. iScience, 25(12), 105527.

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Macrophage-Splenocyte Co-culture for Tularemia

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Co-cultures of bone marrow-derived macrophages (BMDM) and splenocytes were performed as previously described [26 (link)]. Briefly, BMDM were cultured for 6–7 days to a confluent monolayer in 24-well or 48-well plates in Dulbecco’s modified Eagle’s medium (Lonza) supplemented with 10% serum-inactivated fetal bovine serum (Hyclone, Logan, UT), 10 ng/ml mouse M-CSF (PeproTech, Rocky Hill, NJ), 0.2 mM L-glutamine, 0.1 mM nonessential amino acids, 10 mM HEPES buffer, 1 mM sodium pyruvate, and 1 mM sodium bicarbonate. BMDM were infected with F. tularensis LVS at a multiplicity of infection (MOI) of 1:10 (bacterium to BMDM) for 2 hours, after which the monolayer was washed and treated with 50 μg/ml gentamicin for 45 minutes. The cells were then washed, and splenocytes were added to the infected BMDM at a ratio of 1:2 (splenocytes to BMDM) or as indicated in figure legends. After 2 and 3 days, non-adherent cells were recovered and stored in RNAlater (Invitrogen, Waltham, MA) at -80°C for gene expression analyses. Supernatants were also saved at -80°C for cytokine analyses. Remaining BMDM were lysed and plated for CFU as previously described [26 (link)]. Additional cultures were lysed the day of infection to assess bacterial uptake.

Mittereder L.R., Swoboda J., De Pascalis R, & Elkins K.L. (2023). IL-12p40 is essential but not sufficient for Francisella tularensis LVS clearance in chronically infected mice. PLOS ONE, 18(3), e0283161.

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Polarization of Murine and Human Macrophages

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Polyinosinic–polycytidylic acid (poly(I:C)) was purchased from InvivoGen. Mouse IFNβ (#12405-1) was from PBL. Mouse M-CSF (#315-02), mouse recombinant IL-6 (#315-05), mouse recombinant IL-4 (#214-14), human recombinant M-CSF (#300-25-2), human recombinant IL-6 (#200-06), human recombinant IL-4 (#200-04) were from PeproTech. The ELISA kits for mIL-6 (#88-7064-22) and mIL-4 (#88-7044-22) were from eBioscience.
Mouse IL-4-neutralizing antibodies were from Bio-x cell (#BE0045), mouse IL-6-neutralizing antibody (#504512) was from Biolegend. The ERK1/2 inhibitor U0126 (#U120) was from Sigma. SHP099 (#HY-1003881) was from MCE. The NE-PER Nuclear and Cytoplasmic Extraction kit (#78833) was from Thermo Fisher. Primary antibodies for Western blot were purchased from Cell Signaling Technology (arginase-1, #93668; pSTAT6-Y641, #565543; STAT6, #93623; pSTAT3-Tyr705, #9145; STAT3, #9139; STAT1, #14994; Erk1/2, #4695; pErk1/2, #4370), and Genscript (Actin, #A00730), Abclonal (GFP, #AE012). Antibodies for Flow Cytometry were purchased form Biolegend: anti-CD16/32 (#101330), AF488-anti-Ly6C (#128022), BV421-anti-F4/80 (#123132), AF647-anti-CD206 (#141712).

Yang L., Guo P., Wang P., Wang W, & Liu J. (2023). IL-6/ERK signaling pathway participates in type I IFN-programmed, unconventional M2-like macrophage polarization. Scientific Reports, 13, 1827.

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Bone Marrow Macrophage Polarization

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Bone marrow cells from 2 femurs were cultured for 6 days in 30 mL size teflon bags (#PL30, PermaLife, Origen) in DMEM (#21969-035, Gibco, Life Technologies) complemented with 10% FBS (#10270106, Gibco, Thermo Fisher Scientific), 1% l-Glutamine (#25030-024, Gibco, Thermo Fisher Scientific), 1% HEPES 1 mol/L (#H0887, Sigma-Aldrich), 1% penicillin–streptomycin (#15140-122, Gibco, Thermo Fisher Scientific), and 10 ng/mL mouse M-CSF (#14-8983-80, Thermo Fisher Scientific; M0). Media were exchanged every 2 days. For polarization experiments, cells were seeded in Nunc Multidishes with UpCell Surface (#174899, Thermo Fisher Scientific) with 10 ng/mL mouse M-CSF for M0 or 10 ng/mL mouse IFNγ (#315-05, PeproTech) plus 10 ng/mL LPS (#L4391, Sigma) for M1. Polarization was done for 24 hours in the presence or absence of 0.5 μmol/L of bemcentinib (BergenBio, ASA).

Tirado-Gonzalez I., Descot A., Soetopo D., Nevmerzhitskaya A., Schäffer A., Kur I.M., Czlonka E., Wachtel C., Tsoukala I., Müller L., Schäfer A.L., Weitmann M., Dinse P., Alberto E., Buck M.C., Landry J.J., Baying B., Slotta-Huspenina J., Roesler J., Harter P.N., Kubasch A.S., Meinel J., Elwakeel E., Strack E., Quang C.T., Abdel-Wahab O., Schmitz M., Weigert A., Schmid T., Platzbecker U., Benes V., Ghysdael J., Bonig H., Götze K.S., Rothlin C.V., Ghosh S, & Medyouf H. (2021). AXL Inhibition in Macrophages Stimulates Host-versus-Leukemia Immunity and Eradicates Naïve and Treatment-Resistant Leukemia. Cancer Discovery, 11(11), 2924-2943.

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Differentiation of Murine Bone Marrow Macrophages

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Wild type, 2–3 months old female C57/Bl6 mice were purchased from Jackson laboratories. Female Stat6^−/− mice were purchased from Jackson Laboratories. Mice were sacrificed and bone marrow was isolated form the tibiae and femora of the animals. Red blood cell lysis was carried out and cells were plated in differentiation media containing 10% FBS, Dulbecco’s Modified Eagle’s Medium (DMEM) and 20ng/ml mouse M-CSF (Peprotech). On the third day of differentiation, media was replaced with fresh differentiation media. Cytokine treatments, sorting procedures were carried out on the 6^th day of differentiation.

Daniel B., Chen A.Y., Sandor K., Zhang W., Miao Z., Lareau C.A., Yost K.E., Chang H.Y, & Satpathy A.T. (2024). Regulation of immune signal integration and memory by inflammation-induced chromosome conformation. bioRxiv.

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Murine Bone Marrow Macrophage Differentiation

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Wild type, 2–3 months old female C57/Bl6 mice were purchased from Jackson laboratories. All mice were housed in a specific-pathogen-free facility and were used for experiments at 8–12 weeks of age. All experiments were performed according to protocols approved by the Institutional Animal Care and Use Committees of Stanford University (protocol number: 33814). Mice were sacrificed and bone marrow was isolated form the tibiae and femora of the animals. Red blood cell lysis was carried out and cells were plated in differentiation media containing 10% FBS, Dulbecco’s Modified Eagle’s Medium (DMEM) and 20ng/ml mouse M-CSF (Peprotech). On the third day of differentiation, media was replaced with fresh differentiation media. Cytokine treatments, sorting procedures were carried out on the 6^th day of differentiation.

Daniel B., Belk J.A., Meier S.L., Chen A.Y., Sandor K., Czimmerer Z., Varga Z., Bene K., Buquicchio F.A., Qi Y., Kitano H., Wheeler J.R., Foster D.S., Januszyk M., Longaker M.T., Chang H.Y, & Satpathy A.T. (2022). Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state. Molecular cell, 83(1), 121-138.e7.

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Isolation and Co-culture of TAMs, BMDMs, and CD8+ T Cells

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For in vitro co-culture experiments, TAMs (7-AAD^⁻CD45⁺CD11b⁺F4/80⁺) were isolated from advanced PE tumors using a FACSAria II cell sorter (BD Biosciences). and cultured in DMEM medium supplemented with 10% FBS and 10 ng/mL mouse M-CSF (BioLegend, # 576404). 1x10⁵/well TAMs were seeded onto a 48-well plate and allowed to adhere overnight. Bone marrow-derived macrophages (BMDMs) were obtained from FVB/N mice by modifying previously described protocols (15 (link), 16 (link)). Bone marrow cells were seeded on ultra-low attachment plates (Corning) or petri dishes (Falcon) and cultured in DMEM growth medium (DMEM + 10% FBS + 100 μg/mL penicillin–streptomycin) supplemented with 10 ng/mL mouse M-CSF (BioLegend, # 576404). On day 3, cell culture was top-up with fresh DMEM growth medium (same as original volume) with 10 ng/mL M-CSF. Cells were then incubated for another 4 days before harvesting adherent cells (BMDMs). Mouse CD8⁺ T cells were isolated from spleens of FVB mice using a mouse CD8⁺ T cell isolation kit (StemCell, # 19853). CD8⁺ T cells were cultured alone or co-cultured with TAMs or BMDMs at a ratio of 1:1 in RPMI 1640 supplemented with 10% FBS, 10 ng/mL mouse M-CSF, 0.055 mM 2-mercaptoethanol, 2 ng/mL IL-2 (Peprotech), 2.5 ng/mL IL-7 (Peprotech) and 50 ng/mL IL-15 (Peprotech) for 2 days.

Lin Z., Wang Q., Jiang T., Wang W, & Zhao J.J. (2023). Targeting tumor-associated macrophages with STING agonism improves the antitumor efficacy of osimertinib in a mouse model of EGFR-mutant lung cancer. Frontiers in Immunology, 14, 1077203.

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Differentiation of Bone Marrow-Derived Macrophages

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Bone marrow cells from femur exudates of C57BL/6 were grown at 37°C in a humidified incubator in RPMI 1640 medium (RPMI, Invitrogen) containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 20 ng/ml mouse M-CSF (Peprotech, Rocky Hill, NJ, USA) for 7 days. Differentiated BMDMs were replated into six-well dishes 18 h before infection.

Wang X., Sun J., Wan L., Yang X., Lin H., Zhang Y., He X., Zhong H., Guan K., Min M., Sun Z., Yang X., Wang B., Dong M, & Wei C. (2020). The Shigella Type III Secretion Effector IpaH4.5 Targets NLRP3 to Activate Inflammasome Signaling. Frontiers in Cellular and Infection Microbiology, 10, 511798.

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