Human peripheral blood was obtained from healthy volunteers with approval from the institutional review board as described previously.27 (link) Peripheral blood mononucleated cells were isolated from human peripheral blood using Histopaque (Sigma, Germany) following negative isolation with magnetic beads (Stem Cell, Canada), yielding >95% CD14+ monocytes. Cells were cultured in macrophage serum-free medium (Life Technologies, USA) supplemented with Nutridoma SP (Roche, Germany) and penicillin/streptomycin (Sigma, Germany) for 6 days in the presence of 100 ng/mL recombinant human macrophage colony-stimulating factor (Peprotech, USA). Macrophages were exposed to 15 μg/mL oxidized LDL (oxLDL) (Hycultec, Germany) in addition to 100 ng/mL CCL19 or 100 ng/mL CCL21 for 24 hours. The experiment was repeated five times. After washing the cells, we performed Oil Red O staining. For Oil Red O-staining analysis, we used the program ImageJ and the plugin colored convolution for ImageJ (National Institutes of Health). We measured the positively stained area in relation to the total area of the cells for each group. Untreated macrophages served as negative controls.
Macrophage serum free medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Macrophage serum-free medium is a specialized cell culture medium designed to support the growth and maintenance of macrophage cells in vitro. It is formulated to provide the necessary nutrients and growth factors required for the optimal proliferation and differentiation of macrophages without the addition of serum.
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Lab products found in correlation
Nutridoma sp, by Roche (4 mentions)
Fetal bovine serum (fbs), by Cytiva (2 mentions)
M csf, by R&D Systems (2 mentions)
Dioc6 3, by Sony (2 mentions)
Lactate assay kit, by Abcam (2 mentions)
Dioc6 3, by Thermo Fisher Scientific (2 mentions)
Dioc6 3, by IBM (2 mentions)
Spss statistics 22, by IBM (2 mentions)
Sh800 instrument, by Sony (2 mentions)
Fungizone, by Thermo Fisher Scientific (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Human macrophage colony stimulating factor m csf, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Pan monocyte isolation kit, by Miltenyi Biotec (2 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Histopaque, by Merck Group (1 mentions)
Recombinant human macrophage colony stimulating factor, by Thermo Fisher Scientific (1 mentions)
Magnetic beads, by STEMCELL (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
24 well plate, by Thermo Fisher Scientific (1 mentions)
25 protocols using macrophage serum free medium
1
Oxidized LDL and Chemokine Effects on Macrophages
2
Isolation and CpG Stimulation of B Cells
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Peripheral blood mononuclear cells (PBMCs) were purified from healthy donor buffy coats (Department of Transfusion Medicine, Uppsala University Hospital, Sweden) using Ficoll density-gradient centrifugation. The B cells were isolated from PBMCs by positive selection using CD19+ B-cell isolation kit (Miltenyi Biotec) according to manufacturer’s instructions. The cells were cultured in 1 ml volumes in 24-well plates (Nunc) in macrophage serum-free medium (Life Technologies) at the concentration of 3 × 106 B cells/ml. The cells were stimulated with a phosphorothioate-modified CpG A oligonucleotide ODN2216 (CyberGene) at the concentration of 3 μg/ml and incubated for 5 h at 37 °C with 5 % CO2. The harvested cells were stored in RLT buffer (QIAGEN) at −80 °C.
3
Extracellular Matrix Degradation Assay
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For analysis of extracellular matrix degradation, microspheres were generated from a solution of 3% Alginate (Pronova UP MVG alginate, Nova Matrix, Norway), 1 mg/ml of human collagen type I (Advanced BioMatrix, San Diego, California) and 100 µg/ml of DQ collagen (Invitrogen, Paisley, UK). Microspheres were then placed in macrophage serum free medium (Life Technologies) and incubated at 37°C. Fluorescence was read on a GloMax Discover (Promega) at an absorption maxima of 495 nm and fluorescence emission maxima of 515 nm.
4
Isolation and Culture of Bone Marrow-Derived Macrophages
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Bone marrow derived macrophages were isolated and treated as described previously:9 (link) In short, bone marrow was harvested from murine hind legs. Erythrocytes were lysed (150 mM NH4Cl, 10 mM KHCO3, 0.2 mM EDTA). Ficoll-Paque (GE Healthcare) was used for gradient centrifugation. Differentiation medium (DMEM, 30% L929 supernatant, 20% FBS, 1% Pen/Strep) was added on bacterial plates for 5 days. Differentiated macrophages were counted and seeded in macrophage serum free medium (Thermo Fisher Scientific) with 1% Pen/Strep.
5
Phagocytosis Assay of RAW264.7 Cells
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The phagocytic function of RAW264.7 cells was assessed using a Vybrant Phagocytosis Assay Kit (Thermo Fisher Scientific)54 (link). RAW264.7 cells were seeded at 5 × 104 cells/well in 96-well microplates in DMEM supplemented with 10% FBS. After 12 h, the medium was exchanged to Macrophage Serum-Free Medium (Thermo Fisher Scientific), and vehicle or modified SP-A (50 μg/ml) was added to the cells for 2 h. The culture medium was removed, and 100 μl of fluorescein-labeled E. coli bioparticles (5 mg/ml) was added to the cells for 2 h. After the culture medium was removed, trypan blue solution was added to quench the extracellular probe. The fluorescence intensity of each well was then determined with a fluorescence plate reader using 480 nm as the excitation wavelength and 520 nm as the emission wavelength. The phagocytic index was calculated as the fluorescence intensity of the experimental cells relative to the intensity of vehicle-treated cells.
6
Isolation and Culture of Microglia and OPCs
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Microglia were isolated from 3-mo-old adult C57BL/6 mice using a Magnetic-Activated Cell Sorting (MACS) protocol (Fig. 3A ), similar to that described previously (62 (link)). After 48 h, media was changed to macrophage serum-free medium (Thermo Fisher Scientific), containing the antibiotic treatments (SI Appendix, Fig. S2A ). Following a further 48 h, 10 μg/mL myelin debris was added to each well for 4 h.
OPCs were isolated from P6-8 C57BL/6 mouse pups using a MACS protocol (Fig. 3D ), similar to that described previously (62 (link)). Cultures received growth factors for the first 4 d (PDGFα and FGF2), after which antibiotic treatments (SI Appendix, Fig. S2A ) were introduced for a further 6 d (replaced after 3 d) in the absence of growth factors to allow differentiation. Then 10 μM EdU was applied for 3 h prior to fixation.
Standard immunocytochemistry techniques were applied, as described previously (62 (link)). For a full list of antibodies, please seeSI Appendix, Supplementary Materials and Methods .
OPCs were isolated from P6-8 C57BL/6 mouse pups using a MACS protocol (
Standard immunocytochemistry techniques were applied, as described previously (62 (link)). For a full list of antibodies, please see
7
Glioma and GBM Cell Culture Protocol
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GL261 (glioma) cells were obtained from the National Cancer Institute (Frederick, MD, USA). Cells were cultured in macrophage serum-free medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum, 1% penicillin/streptomycin and 1 mM L-glutamine. GBM14 cells have been described previously,18 (link) and they were obtained by Dr J Sarkaria at Mayo Clinic (Rochester, MN, USA) and cultured in StemPro media (Thermo Fisher Scientific), as directed by the manufacturer.
8
Microglial Isolation and Activation
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Microglial cells were isolated from control and Mfp2−/− pups at postnatal day 8 (P8) using magnetic-activated cell sorting (MACS) according to the manufacturer’s instructions (Miltenyi Biotec). Cells were plated in 12-well plates in a Macrophage Serum Free Medium (Thermo Fisher) and stimulated for 24 h with 50 ng/ml IL1β and 20 ng/ml IFNγ or 50 ng/ml IL-4 (all from R and D) to induce a pro- and anti-inflammatory phenotype, respectively.
For confirming recombination of the Mfp2 gene, microglia were isolated from 11-month-old control and Cx3cr1-Mfp2−/− mice. Mice were anesthetized with a mix of Domitor (1 mg/kg) and Nimatek (75 mg/kg) and perfused with approximately 20 ml ice-cold HBSS (without calcium and magnesium). Subsequently, brains were removed and dissociated to a single cell suspension using the Neural Tissue Dissociation Kit (P) according to the manufacturer’s instructions for the automated dissociation using the gentleMACS Dissociator (Miltenyi Biotec). Next, myelin was removed by 22% Percoll gradient and the cell suspension was further processed for microglia separation. Microglia (positive fraction) were separated from other brain cells (negative fraction) by MACS following the manufacturer’s instructions using CD11b MicroBeads (Miltenyi Biotec). Both the positive and negative fractions were further processed for qRT-PCR.
For confirming recombination of the Mfp2 gene, microglia were isolated from 11-month-old control and Cx3cr1-Mfp2−/− mice. Mice were anesthetized with a mix of Domitor (1 mg/kg) and Nimatek (75 mg/kg) and perfused with approximately 20 ml ice-cold HBSS (without calcium and magnesium). Subsequently, brains were removed and dissociated to a single cell suspension using the Neural Tissue Dissociation Kit (P) according to the manufacturer’s instructions for the automated dissociation using the gentleMACS Dissociator (Miltenyi Biotec). Next, myelin was removed by 22% Percoll gradient and the cell suspension was further processed for microglia separation. Microglia (positive fraction) were separated from other brain cells (negative fraction) by MACS following the manufacturer’s instructions using CD11b MicroBeads (Miltenyi Biotec). Both the positive and negative fractions were further processed for qRT-PCR.
9
Evaluating Neutralizing Capacity of GMAb
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Neutralizing capacity of GMAb was evaluated using TF-1 cells as described previously4 (link),27 (link). Briefly, TF-1 cells (40,000 cells/well) were incubated at 37 °C under 5% CO2 in a flat bottom microtiter plates for 3 days in Macrophage Serum Free Medium (Thermo Fisher Scientific, Waltham, MA, USA) containing recombinant human GM-CSF (0.5 ng/ml) with various concentrations of GMAb (0–125 ng/ml). TF-1 cells survival was evaluated as formazan formation using the Cell Counting Kit-8 (Doujindo, Kumamoto, Japan), measuring the absorbance at 450 nm by using a microplate reader (Bio-Rad).
10
Establishment of Cell Lines and Virus Stocks
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Human embryonic kidney 293 (HEK), human leukemia monocytic cell line (THP-1), human rhabdomyosarcoma (RD), and SV40 T-antigen transformed HEK293 (293T) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and maintained following the manufacturer’s instructions. To generate monocyte-derived macrophages (MDMs), isolated CD14+ monocytes were plated in 10 cm Petri dishes at 1.0 × 107 cells/dish. Monocytes were stimulated with 25 ng/mL M-CSF (R&D Systems, Minneapolis, MN) in macrophage serum-free medium (Thermo Fisher Scientific, Waltham, MA) for seven days. MDMs were then maintained in DMEM (Invitrogen, Thermo Fisher Scientific,) containing 10% FBS (HyClone Laboratories, Logan, UT), 25 mM HEPES (Quality Biology, Gaithersburg, MD), and 5 μg/mL Gentamicin (Thermo Fisher Scientific) before use in experiments. The HSV-1 McKrae strain was kindly provided by Dr. Jeffrey I. Cohen (National Institute of Allergy and Infectious Diseases/National Institutes of Health, Bethesda, MD). Virus stock was prepared using Vero cells (ATCC), and virus titer was determined by plaque-forming assay (Blaho et al., 2006 (link)). Sendai virus (SeV) was obtained from Advanced Biotechnologies, Inc. (Eldersburg, MD). HSV-1 was used at an MOI of 1, and SeV was used at a final concentration of 40 HA units/mL.
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