Receptor-associated protein (RAP) was expressed as a GST fusion protein in bacteria and purified as described (Herz et al., 1991 (link)). LPS, serotype 055.B5, and Tamoxifen (TAM) were from Sigma-Aldrich. Recombinant mouse CSF-1 and Quantikine ELISA kits were purchased from R&D systems. All primers and probes for RT-qPCR experiments were from Applied Biosystems.
Recombinant mouse csf 1
Manufactured by R&D Systems
Recombinant mouse CSF-1 is a hematopoietic growth factor produced in a mouse myeloma cell line. It is a glycoprotein that stimulates the survival, proliferation, and differentiation of mononuclear phagocytes, including monocytes and macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Elements software, by Nikon (2 mentions)
Eclipse ti e inverted microscope, by Nikon (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Lsr 2, by BD (2 mentions)
Celltracker green cmfda dye, by Thermo Fisher Scientific (2 mentions)
Recombinant mouse csf 2, by R&D Systems (2 mentions)
Allophycocyanin tagged secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Tamoxifen tam, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Benzonase, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Bovine serum, by R&D Systems (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Recombinant mouse rankl, by R&D Systems (1 mentions)
Rabbit anti ki67 antibody, by Cell Signaling Technology (1 mentions)
Lps from e coli serotype 055 b5, by Merck Group (1 mentions)
C12 ie dap, by InvivoGen (1 mentions)
12 protocols using recombinant mouse csf 1
1
Bacterial Expression and Purification of RAP
2
Live Cell Imaging of Tumor Cells
3
Stimulation of RAW264.7 Macrophages with CSF1
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RAW264.7 cells (106) were seeded into cell culture dishes 6 cm in diameter and incubated in 4 mL RPMI/10% fetal bovine serum (Biochrom) for 24 h at 37 °C in a humidified 5% CO2 atmosphere. Cells were washed once with phosphate-buffered saline (PBS) and starved in 3 mL RPMI/0.1% bovine serum albumin for another 24 h. Cells were then treated for 1 h at 37 °C with drugs at the indicated final concentrations, with 0.5% DMSO as vehicle control, or left untreated, and finally stimulated for 2 min at 37 °C with 100 ng/mL recombinant mouse CSF1 (R&D Systems) by adding 1 mL prewarmed starving medium containing 400 ng/mL CSF1. Dishes were placed on ice and cells were immediately washed with ice-cold PBS and lysed with 50 µL lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 0.5% desoxycholate, 0.2% sodium dodecyl sulfate, 1 mM ethylene glycol-bis[β-aminoethyl ether]-N,N,N′,N′-tetraacetic acid, 100 mM NaF, 10 mM Na4P2O7, 1 mM Na3VO4, 1% Phosphatase Inhibitor Cocktail 3 [Sigma-Aldrich], 1 mM phenylmethyl sulfonyl fluoride, Complete Protease Inhibitor Cocktail [Roche CustomBiotech; 1 tablet/10 mL], and 50 U/mL Benzonase [Sigma-Aldrich]). Lysed cells were scraped into Eppendorf tubes, incubated on ice for 20 min, and centrifuged at 4 °C for 10 min at 13,000 rpm; supernatants (lysates) were transferred into new tubes and stored at −80 °C.
4
Live Cell Imaging of Tumor Cells
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For live imaging experiments, tumor cells were plated at a density of 1 × 105 per well in a 12-well glass-bottom plate (In Vitro Scientific), either alone, with an equal number of CAG-EGFP BMDMs, or with BMDM-CM. In experiments with BMDMs, the media were supplemented with 10 ng/mL recombinant mouse CSF-1 (R&D Systems). Twenty-four hours after plating, cells were treated and imaged for 24 hr using a Nikon Eclipse TiE inverted microscope equipped with an environmental chamber maintained at 35°C–37°C and 5% CO2. Mitotic arrest duration in live imaging movies was measured using Nikon Elements Software (Nikon) with at least 30 cells analyzed per treatment condition per experiment.
Additional information on experimental methods can be found in theSupplemental Experimental Procedures .
Additional information on experimental methods can be found in the
5
Isolation and Differentiation of Murine Bone Marrow-Derived Macrophages
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We used male and female animals of 6-10 weeks of age. BMDMs were extracted from C57BL/6 mice using standard protocols (Gocheva et al., 2010 (link)). Following euthanasia, femurs and tibiae were harvested from both legs of the mice under sterile conditions. After careful cleaning of the tissue, the bone marrow was flushed using a 25-gauge needle and passed through a 40 μm strainer. The bone marrow flush was cultured in low-attachment culture dishes (VWR, 25384-342) in high-glucose DMEM supplemented with 10% FBS, 10 ng/ml Recombinant Mouse CSF-1 (R&D Systems, 416-ML), and 1× Antibiotic-Antimycotic (Gibco, 15240) for 7 days. Medium was replaced every other day to induce macrophage differentiation. All protocols involving animal work complied with relevant regulatory standards and were approved by New York University’s University Animal Welfare Committee (UAWC protocols 17-1496 and 19-1515).
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Generating Macrophages from Murine Bone Marrow
7
Cat Osteoclast Culture Protocol
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The protocol was modified from a published rodent osteoclast culture protocol38 . The long bones were dissected from cats at post mortem and cut off at the proximal and distal ends. Bone marrow was flushed out in α-MEM containing 10% FBS followed by red cell lysis in Lysis Buffer (150 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA) for 5 min, on ice. The bone marrow cells were resuspended in α-MEM containing 10% FBS, 2 mM L glutamine, 100 IU/ml benzyl penicillin, 100 mg/ml streptomycin, and CSF-1 (recombinant mouse CSF-1, 10 ng/ml, R&D Systems) and plated on a T75 flask for 24 h at 37 °C in 5% CO2/95% air. On the following day, non-adherent cells were collected and plated on a dentine disc in each well of a 24 well plate (dentine discs were a generous gift from Professor Timothy R. Arnett, University College London, London, UK) or Corning Osteo Assay Surface multiple well plates (1 × 105 cells/cm2) with CSF-1 (10 ng/ml) at 37 °C in 5% CO2/95% air. On day 4, 90% of the medium was changed and, replaced with a new α MEM containing CSF-1 (10 ng/ml) and RANKL (recombinant mouse RANKL, 3 ng/ml, R&D Systems). The culture media was subsequently changed twice weekly for up to 10–14 days.
8
Microglia Proliferation Assay with CSF1 and GW2580
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Primary microglia were seeded at a density of 1x105 cells per well in a 96-well plate in DMEM supplemented with 10% FBS, 100U pen/strep and glutamax. To determine the effect of CSF1 and GW2580 on cell proliferation, microglia were treated with recombinant mouse CSF1 (0-30ng/ml; R&D Systems; Cat No. 416-ML-10) for 48h with increasing concentrations of GW2580 (0-5μM). After treatment, cells were fixed with 4% paraformaldehyde for 20 min. at room temperature, washed with PBS then blocked and permeabilized in PBS containing 0.3% Triton-X 100 (PBST) and 5% goat serum for 1h at room temperature. Next, the cells were incubated with a rabbit anti-Ki-67 antibody (Dilution 1:200; Cell Signaling; Cat No. 9129S) overnight at 4°C. After washing with PBS, cells were incubated with Alexa Fluor 488 conjugated goat anti-rabbit IgG diluted 1/1000 in PBST for 1h. (ThermoScientific). After washing with PBS, proliferation was determined by counting the number of microglia per well, as well as the number of Ki-67+ cells per well using ImageJ (NIH).
9
Intracellular Flow Cytometry for DNA Content Analysis
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For intracellular flow cytometry and DNA content analyses, cells were plated at a density of 1 × 105 per well in a 12-well plate, either alone, with an equal number of stromal cells, or BMDM CM. For co-cultures only CAG-EGFP BMDMs were used, and other stromal cell lines were fluorescently labeled with CellTracker Green CMFDA Dye (Molecular Probes). In experiments with BMDMs, the media were supplemented with 10 ng/mL recombinant mouse CSF-1 (R&D Systems). In experiments with MHCII+ BMDMs, the media were supplemented with 10 ng/mL recombinant mouse CSF-2 (R&D Systems). Twenty-four hours after plating, cells were treated and then collected at indicated time points by trypsinization. The samples were then fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and stained overnight at 4°C using primary antibodies at the concentrations listed in Table S2 . Allophycocyanin-tagged secondary antibodies (Jackson ImmunoResearch) were used at a 1:300 dilution and incubated for 1 hr at room temperature, followed by resuspension in FACS buffer containing DAPI (5 μg/mL) for DNA content evaluation. For analysis, samples were run on a BD LSR II (Becton Dickinson), and all subsequent compensation and gating were performed with FlowJo analysis software (Tree Star).
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Intracellular Flow Cytometry for DNA Content Analysis
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