Bone marrow cells were isolated from tibias and femurs of WT mice and grown in complete medium (RPMI-1640 medium containing 10 mM glucose, 2 mM L-glutamine, 100 U ml–1 penicillin-streptomycin and 10% FCS) with 20 ng ml–1 murine macrophage colony- stimulating factor 1 (CSF-1; Peprotech) for 7 days, and supplemented with CSF-1 on days 3 and 5. On day 7 macrophages were harvested and then maintained in 20 ng ml–1 CSF-1 for subsequent experiments in which they were either maintained in medium alone (M0), or stimulated with 50 ng ml–1 IFN-γ (R&D systems) and 20 ng ml-1 LPS (M1), or 20 ng ml–1 IL-4 (M2) (Peprotech) for 18 h.
Csf 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CSF-1 is a laboratory instrument designed for the separation and purification of specific molecules or cells from complex biological samples. It utilizes centrifugal force to separate components based on their density and sedimentation properties. The core function of the CSF-1 is to enable efficient sample preparation for downstream analysis or experimentation, but its exact intended use may vary depending on the specific application.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Csf 1, by R&D Systems (5 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Il 4 m2, by Thermo Fisher Scientific (2 mentions)
Ifn γ, by R&D Systems (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Recombinant human csf 1, by Thermo Fisher Scientific (2 mentions)
Recombinant human rankl, by Thermo Fisher Scientific (2 mentions)
Il 34, by Thermo Fisher Scientific (2 mentions)
Ultra low attachment flask, by Corning (2 mentions)
Mouse seroblock fcr, by Bio-Rad (1 mentions)
34 protocols using csf 1
1
Differentiation of Mouse Bone Marrow-Derived Macrophages
2
Modulating Monocyte Differentiation
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To further analyze the role of CD115 signaling during monocyte differentiation, 100 ng/mL of their ligands, CSF1 and IL34 (both from Peprotech), was added independently or in combination to the culture of mDC and tolDC on day 0 and day 4. Phenotype and functional characterization of CSF1- and/or IL34-treated cells were analyzed on day 6.
3
Macrophage differentiation and polarization
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Bone marrow cells were grown in complete medium (RPMI-1640 medium containing 10 mM glucose, 2 mM l -glutamine, 100 U ml–1 penicillin–streptomycin and 10% FCS) with 20 ng ml−1 murine macrophage colony- stimulating factor 1 (CSF-1; Peprotech) for 7 days, and supplemented with CSF-1 on days 3 and 5. On day 7, macrophages were harvested and then maintained in 20 ng ml−1 CSF-1 for subsequent experiments in which they were either maintained in medium without any further additions (M0), or stimulated with 50 ng ml–1 IFN-γ (R&D systems) and 20 ng ml−1 LPS (M(γ + LPS)), or 20 ng ml−1 IL-4 (Peprotech; MI(L-4)), for 18 h. In some experiments, cells were treated with 50 μM T863 (Sigma) and/or 10 μM PGE2 (Sigma) throughout the 18 h period.
4
Characterization of Macrophage Phenotypes
5
Protein Extraction and Western Blotting
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The extraction of protein and Western blotting were performed as described previously.32 In short, lysis of specimens was carried out on ice for 0.5 hour in the assay buffer for radio immunoprecipitation from KenGEN (China). Centrifugation of lysates was performed at 14 000 × g and 4℃ for 15 minutes. After collecting the supernatant, concentration of protein was estimated using the bicinchoninic acid assay kit from Pierce (Rockford, USA). After separating equal amounts of protein on 10% SDS‐PAGE by electrophoresis, their transfer was performed onto PVDF (polyvinylidene difluoride membranes) from Millipore Corporation (USA) and blocked for 2 hours. Then, they were incubated overnight with a primary antibody, and then with respective secondary antibodies, and visualized using ECL (enhanced chemiluminescence reagents). The primary antibodies are as follows: Cyclin D1 (55 506, Cell Signaling Technology), CDK4 (12 790, Cell Signaling Technology), β‐acitn (A5441, Sigma) and CSF‐1 (TA806568, Thermo Fisher, USA).
6
Macrophage Differentiation and Fatty Acid Treatment
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C57BL/6 (Strain 000664) and Ppara−/− knockout (Strain 008154) mice were obtained from Jackson Laboratory (Farmington, CT). Bone marrow was isolated from 8-10 week old C57BL/6 and Ppara−/− knockout mice by the St. Jude Children’s Research Hospital Animal Resources Center, and macrophages were derived from the bone marrow using the method established by Toda et al. (2021) (link). Cells (4.2 x 107) were seeded in 6-well tissue culture plates and grown in DMEM and 10% fetal bovine serum with 1 ng/ml CSF-1 (ThermoFisher) at 37°C 5% CO2. On day 6, bone marrow-derived macrophages (BMDM) were washed with phosphate buffered saline and then changed to media containing 1% dimethyl sulfoxide (DMSO) ± fatty acid treatment and incubated 20-24 h. RAW 264.7 mouse macrophage cells (ATCC number TIB-71) were grown in DMEM plus 10% fetal bovine serum containing 1% DMSO ± fatty acid at 37°C 5% CO2.
7
Quantitative Real-Time PCR Protocol
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The PCR reactions were performed based on Taq-Man chemistry using the probes specific for the following genes: Csf1 (Mm00432686_m1), Ccr6 (Mm99999114_s1), Cxcr4 (Mm01996749_s1), Drg1 (Mm00492246_m1), Foxo1 (Mm00490672_m1), Nf2 (Mm00477771_m1), Nedd9 (Mm01324843_m1), Mmp14 (Mm00485054_m1), Spp1 (Mm00436767_m1), Flt1 (Mm00438980_m1), Plaur (Mm01149438_m1), Tgfb1 (Mm01178820_m1), Pgk1 (Mm00435617_m1), and Rpl13a (Mm01612987_g1) (all Thermo Fisher Scientific, Waltham, MA, USA). Each amplification cycle was performed at 95 °C for 15 s and at 60 °C for 1 min (total 40 cycles) in ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Twenty-five nanograms of cDNA were used for a single reaction and each sample was prepared in triplets (technical repetition). The relative quantification level of examined gene expression, referred to as fold change, was calculated based on differences in ΔΔCt values of the studied genes in relation to control housekeeping genes (Pgk1 or Rpl13a) by using DataAssist 3.01 software (Thermo Fisher Scientific, Waltham, MA, USA).
8
Establishing FL-FDC and FL-Macrophage Co-Cultures
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FL-FDC co-cultures were established with HK cells, a non-immortalized FDC cell line generated from normal tonsils which was kindly provided by Dr. Yong Sung Choi [20 (link)] at a 1:20 ratio (HK:FL), as previously described [21 (link)]. This cell line has been widely used to mimic lymphoma - LN stroma interaction [22 (link)–24 (link)].
FL-macrophage co-cultures were established at a 1:4 ratio (Mϕ:FL). Macrophages were derived from peripheral blood mononuclear cells (PBMCs) of healthy donors (Banc de Sang i Teixits, Barcelona). Blood samples were enriched in the monocyte population using RosetteSep (Human monocyte enrichment cocktail) (STEMCELL Technologies, Grenoble, France) and then cultured for 7 days with 100 ng/mL CSF-1 (Thermo Fisher Scientific, Waltham, MA, USA). When specified, differentiated macrophages were polarized to M1 (20 ng/mL IFNγ (Gibco, Thermo Fisher Scientific) + 100 ng/mL LPS (Sigma-Aldrich, St. Louis, MO)) or M2 (20 ng/mL IL4 (PeproTech, Rocky Hill, NJ)) for 24 h.
FL-macrophage co-cultures were established at a 1:4 ratio (Mϕ:FL). Macrophages were derived from peripheral blood mononuclear cells (PBMCs) of healthy donors (Banc de Sang i Teixits, Barcelona). Blood samples were enriched in the monocyte population using RosetteSep (Human monocyte enrichment cocktail) (STEMCELL Technologies, Grenoble, France) and then cultured for 7 days with 100 ng/mL CSF-1 (Thermo Fisher Scientific, Waltham, MA, USA). When specified, differentiated macrophages were polarized to M1 (20 ng/mL IFNγ (Gibco, Thermo Fisher Scientific) + 100 ng/mL LPS (Sigma-Aldrich, St. Louis, MO)) or M2 (20 ng/mL IL4 (PeproTech, Rocky Hill, NJ)) for 24 h.
9
Osteoclastogenesis Induction and Regulation
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CSF-1 and ODF induced osteoclasts were obtained from R&D Systems Inc., USA. The bone marrow cells were cultured in Modified Eagle’s medium (Thermo Fisher USA) supplemented with fetal bovine serum (FBS) (10%) (Thermo Fisher USA), penicillin (1%), streptomycin (1%) and CSF-1 (10 ng/ml) for 24 h. Bone marrow monocytes (BMMs) were subjected to culture in Modified Eagle’s medium supplemented with FBS (10%), CSF-1 (30 ng/ml) and ODF (50 ng/ml). The TIB-71 macrophage cells (ATCC USA) were cultured in Dulbecco’s Modified Eagle’s medium (Thermo Fisher USA) supplemented with FBS (10%), streptomycin and penicillin (1%). The TIB-71 cells were transfected with ODF (50 ng/ml) for osteoclastogenesis. The cells were incubated under controlled conditions with 95% humidity and 5% CO2. The medium was replaced with a fresh one after every 48 h. The TIB-71 cells were transfected with miR-23b-3p mimics, anti-miR-23b-3p and negative control (NC) using Lipofectamine 2000 reagent (Thermo Fisher USA) for 5 days (mimics and anti-miR) and 2 days (NC). For blocking the expression of PI3K, the TIB-71 cells were treated with PI3K inhibitor LY-294,002 hydrochloride (40 mM) (Sigma-Aldrich USA), also called an autophagy inhibitor, for 24 h.
10
Workflow for Bone Marrow Macrophage Isolation and Brain Immune Cell Analysis
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BM was collected from femurs and tibias as described before (Ulland et al., 2017 (link)). To prepare BMM for antibody binding, survival, and immunoprecipitation assays, BM cells were counted and plated at 10 × 106 cells/100-mm Petri dish in RPMI supplemented with Glutamax, penicillin/streptomycin, nonessential amino acids, pyruvate, 10% heat-inactivated fetal bovine serum (complete RPMI) and 50 ng/ml CSF-1 (Peprotech). Cells were cultured for 7 d before use.
To perform scRNA-seq of the immune cells in brain, whole brains of 22-wk-old (5.5 mo) mice from AL002c- or control IgG-treated groups were dissected after transcardial perfusion with ice-cold PBS and dissociated as previously described (Song et al., 2018 (link)). Immune cells were recovered after Percoll (GE) separation, as described (Mildner et al., 2007 (link)).
To perform the biochemical analysis of brain after AL002c chronic treatment, brains were removed after transcardial perfusion with ice-cold PBS. Cortex and hippocampus were dissected out from left brain hemispheres and flash frozen; right brain hemispheres were fixed in 4% PFA for 2 d at 4°C and rinsed in PBS before incubation for 2 d at 4°C in 30% sucrose. Dehydrated brain tissues were frozen in a 2:1 mixture of 30% sucrose and optimal cutting temperature compound for obtaining serial 40-µm cryosections.
To perform scRNA-seq of the immune cells in brain, whole brains of 22-wk-old (5.5 mo) mice from AL002c- or control IgG-treated groups were dissected after transcardial perfusion with ice-cold PBS and dissociated as previously described (Song et al., 2018 (link)). Immune cells were recovered after Percoll (GE) separation, as described (Mildner et al., 2007 (link)).
To perform the biochemical analysis of brain after AL002c chronic treatment, brains were removed after transcardial perfusion with ice-cold PBS. Cortex and hippocampus were dissected out from left brain hemispheres and flash frozen; right brain hemispheres were fixed in 4% PFA for 2 d at 4°C and rinsed in PBS before incubation for 2 d at 4°C in 30% sucrose. Dehydrated brain tissues were frozen in a 2:1 mixture of 30% sucrose and optimal cutting temperature compound for obtaining serial 40-µm cryosections.
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