For Research Use Only. June 30th, 2016 A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. With regards to supplementation with M‐CSF during the differentiation, we used 80 ng/ml for both monocyte induction from day 9 to day 14 or 15 and later, the macrophage differentiation from monocytes. 1% FBS (i.e., no Triton). Discard supernatant. Transfer the cells to a 15-mL tube. Permeabilize the cells with this solution for 15 min. Add FITC-conjugated anti-Mac-1 and APC-conjugated anti-4/80 antibodies (both diluted 1:400) to the cells. Replace media in culture dish with the prepared media containing M-CSF and IL-4 (if using). cells to be used for phagocytosis assays for 7 d. 14. 17. Add 2 mL of additional trypsin. 12. The addition of IL-4 or IL-10 to M-CSF – leading to differentiation into M2a or M2c MDM, respectively - did not result in changes in macrophage morphology (Figure 1C). numerous types of experimental manipulations, including morphological, gene expression, and physiological studies. Equilibrate the Mouse Macrophage Nucleofector Solution to room temperature. Prepare complete RPMI 1640 medium by supplementing RPMI 1640 medium with fetal bovine serum to a final concentration of 10%, 2 mM L-glutamine (if using medium not currently supplemented with GlutaMAX). Don't have an account ? Incubate the samples for 5 min in 23. Remove the medium from the BMMs (from Step 36). 47. Copyright © 2021 by Cold Spring Harbor Laboratory Press. Incubate the samples in a humidified 37°C, 5% CO2 incubator for 1 h. 20. Additionally, macrophages are specialized cells that carry out numerous tasks in the immune system such as phagocytosis, Calculate the number of cell divisions using the Pass the cells through a cell strainer. Search Our results suggest that the efficiency of GM‐CSF and M‐CSF to drive Mϕs toward M1/M2 phenotypes includes priming effects on eicosanoid biosynthesis, which can affect the final shift toward inflammatory or resolving functions. Protocol for the Generation of M1 or M2a Activated Macrophages Generate monocyte-derived macrophages (MDM) from isolated monocytes by culturing the cells in ImmunoCult™SF Macrophage Differentiation Medium (ImmunoCult™ SF Macrophage Medium Catalog #10961 with added Human Recombinant M-CSF Catalog #78057). Stain the cells with this solution for 30 min. 50. Differentiation into ECs caused a … Start the macrophage differentiation (day 0). RNAs (siRNAs) into these hard-to-transfect cells is described. Problem: CFSE emission is too bright for FACS analysis. Wash the cells twice in PBS containing 1% FBS. The BMMs represent a tractable system to assay these functions in We describe a test for the phagocytic efficiency Adjust accordingly for other dish sizes. to add for a multiplicity of infection (MOI) of 10. Resuspend the cell pellet in an appropriate volume of flow cytometry staining buffer or buffer of choice, and perform a cell count and viability analysis. 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Differentiate cells in a humidified incubator with 5% CO2 at 37°C. of growth factors. The protocol involves several key steps: (1) expansion of iPSCs in feeder-free conditions using chemically defined medium, (2) adapting iPSCs in feeder-dependent culture, (3) formation of three germ layers (ectoderm, mesoderm, and endoderm) containing embryoid bodies (EBs), (4) generation of myeloid precursor cells from EBs in the presence of IL-3 and M-CSF, and (5) terminal differentiation of … It is one of the three experimentally described colony-stimulating factors. Add 500 μL of the plated medium (from Step 42) to the cuvette. Antibody, anti-mouse 4/80 antigen, allophycocyanin (APC)-conjugated (eBioscience), Antibody, anti-mouse CD16/32 (Fc block) (eBioscience), Antibody, anti-mouse Mac-1, fluorescein isothiocyanate (FITC)-conjugated (eBioscience), Celltrace CFSE Cell Proliferation Kit (Molecular Probes), DAPI (4′,6-diamidino-2-phenylindole) (Molecular Probes), Dimethyl sulfoxide containing 5 mM carboxyfluorescein succinimidyl ester (CFSE stock). Bring medium to 37°C. Flow chart of isolation and examples of applications for BMMs. Add 10 mL 10 mM EDTA to each culture dish, and let sit for 10 minutes, or until cells dissociate from dish at room temperature. The latter combination is often used to differentiate monocytes to dendritic cells. Cells are ready to harvest when cells exhibit more granules in the cytoplasm and are a bit elongated. However, little is known about the composition of L929 cell-conditioned media (LCCM) and how it affects the BMDM phenotype. Wash cells with PBS. For a pure macrophage culture, we recommend that you add factors such as M-CSF. that can be used to track cell proliferation. The efficiency of the differentiation is assessed using fluorescence-activated Yanagimachi13 developed a mono et al - 20 ml per T-75 flask and incubate for 6 days at 37°C and 5% CO 2 without medium change. 500 μL PBS. Process BMMs as required for assays for phagocytic activity (Steps 15–28), nucleic acid or protein extraction (Steps 29–30), Problem: BMMs are difficult to trypsinize. 39. Make sure the transfection solution Wash cells twice with PBS every 2–3 d, and add fresh BMM medium. Tissue macrophages can be derived from monocytes. In this protocol, bone marrow cells are grown in culture dishes in the presence of M-CSF, which is secreted by L929 cells and is used in the form of L929-conditioned medium. 10. 19. Terms of Service. Prepare lymphocyte medium containing 20% FBS. Effect of differentiation in M-CSF, GM-CSF, or GM-CSF plus IL-4. Extract RNA, DNA or protein according to the needs of the subsequent experiment. Wash with PBS, spin down at 1,200 rpm for 5 min at RT PBMC or monocyte differentiation to macrophage and polarization (Dr. Muredach Reilly lab by Hanrui Zhang) ... RPMI-1640 and 20% FBS with 100 ng/ml Human M-CSF (Peprotech) (1 vial of M-CSF aliquot with 25 ... Differentiation: D0: 1. Mix gently. (D) Transfection studies. When isolated from blood and cultured in media with serum, adherent monocytes will differentiate into macrophages. T25 or T75 sterile flasks with vented caps (e.g., Using aseptic techniques under sterile conditions, isolate PBMC from whole blood (See. 2003; Doyle et al. 8. All steps are performed at 4°C in the dark. Wash the strainer with another 5 mL of lymphocyte medium. covers the bottom of the cuvette, and avoid air bubbles. 1. Add 6 mL of lymphocyte medium. We investigated and compared a panel of polarization protocols of blood-derived monocytes to achieve a … 12-well plate. Adding other factors including IL-4, IL-10, or TGF-β can help improve viability. Transfection of BMMs using an Amaxa Nucleofector system results in 40%-50% transfection efficiency. can be grown up to three weeks without noticeable cell death or altered morphology. Wash the bioparticles twice, 1 mL of PBS each wash, by centrifugation at 1250g for 15 min. Fix cells in freshly prepared 4% PFA for 20 min. Incubate for 20–25 min at 37°C with occasional swirling. Centrifuge the cells at 300g for 5 min. into a homogenous population of mature BMMs. Under these conditions, the bone marrow monocyte/macrophage progenitors will proliferate and differentiate Normally, CFSE emission is too bright the same day or even the day after. Sterilize the abdomen and hind legs with 70% ethanol. Centrifuge at 200g for 10 min. Recombinant human M-CSF can regulate the proliferation, differentiation, and survival of monocytes, macrophages, osteoclasts and their hematopoietic progenitors . 7. Incubate the cells in a humidified incubator with 5% CO2 at 37°C for 24–48 h. The cells can now be used for subsequent experiments. BMMs are homogenous, have a proliferative capacity, are transfectable, and have a lifespan longer than a week. Also, a method to deliver DNA or small interfering Incubate for 30 6. 27. Adjust the concentration to 2 × 106 cells/mL in BMM medium. Count harvested BMMs (from Step 36). 34. Under these conditions, the bone marrow monocyte/macrophage progenitors will proliferate and differentiate into a homogenous population of mature BMMs. 42. Macrophages are double-positive for Mac-1 and 4/80. Swirl the dish briefly to mix. 32. Cut the bones at both ends to free them. Resuspend the cells by pipetting. Analyze the cells using a fluorescence microscope. Replace with fresh BMM medium. Wash in H2O. To investigate the effect of IL-3/G-CSF or IL-3/M-CSF on hematopoietic in vitro differentiation of PSCs, we established a four-step differentiation protocol (Figure 1A) utilizing human iPSCs (hiPSCs) previously generated from nonmobilized peripheral blood (PB)-derived CD34 + cells (hCD34iPSC11 and hCD34iPSC16) (Ackermann et al., 2014, Lachmann et al., 2014) or the human ESC … Air-dry briefly. Harvest bone marrow in a laminar flow hood. Grow Preincubate the plates in a humidified incubator with 5% CO2 at 37°C. 26. 24. 35. Add 4 mL Trizol directly to a 15-cm dish of BMMs. Adding other factors including IL-4, IL-10, or TGF-β can help improve viability. phagocytic cells such as macrophages have a unique ability to ingest microbes. Add 5 μL of fluorescently labeled zymosan A bioparticles and 5 μL of opsonizing reagent to a 1.5-mL reaction tube containing Briefly vortex the opsonized bioparticles. Monocytes derived from patients with chronic inflammatory diseases could be induced to be anti-inflammatory using this protocol. One possible reason may be the initial CSF used in differentiation protocols. Macrophage colony‐stimulating factor (M‐CSF) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) are not only important hematopoietic growth factors but also potent cytokines that are needed for cell survival, proliferation, differentiation and activation 14. Discard the supernatant and rinse cells with 1X PBS. Then, M-CSF was added at a final concentration of 50ng/ml. (C) Gene expression analyses. Monocytes are highly abundant circulatory effector cells and play a vital role in driving or resolving inflammatory processes depending on their activation phenotype. For example, Clip outward to expose the hind legs. Centrifuge the cells at 300–400 x g for 4–5 minutes at 2–8°C. To generate BMDMs, the myeloid progenitor cells isolated from the bone marrow are cultured in the presence of macrophage colony-stimulating factor (M-CSF), which signals through its receptor M-CSFR (= c-Fms) to mediate monocytic lineage proliferation and differentiation[2, 3]. the proliferation and differentiation of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Calculate the number of bioparticles required Eukaryotic cells also produce M-CSF in order to combat intercellular viral infection. Grow cells in a humidified incubator with 5% CO2 at 37°C for 7 d. 3. Filter through a 0.45-μm filter. Wash the BMMs four times with cold PBS. Add 4 mL of room-temperature trypsin. M1 and M2 macrophage receptor expression is higher in RPMI 10% FBS compared to XVivo 10 Dilute blood sample at least 1:1 with PBS in a conical tube. macrophage (M1) stimulation and significantly suppressed T-cell proliferation. Copyright © 2021 by Cold Spring Harbor Laboratory Press. Flush the bones with lymphocyte medium using a 5-mL syringe and a 25-gauge needle. Wash twice with PBS. M-CSF is known to stimulate differentiation of hematopoietic stem cells to monocyte-macrophage cell populations in culture. Centrifuge cells at 300–400 x g for 4–5 minutes. CFSE is a nonfluorescent cell-permeant. culture dish, 1 mL per well for 24-well or 2 mL per well for 12-well plates (for phagocytosis assays). 22. Make an incision in the midline of the abdomen. Differentiation of Macrophage Subtypes. Macrophage progenitors adhere to the cell dish and are not washed away. The zymosan A bioparticles M1 and M2 macrophage differentiation was performed in each of the base media with cytokines from the CellXVivo ™ M1 and M2 Macrophage Kits added to each media condition in parallel. to macrophage differentiation, van Wilgenburg et al12 estab-lished an embryoid bodies (EBs)-based protocol for IPSDM differentiation in which hematopoiesis was induced under serum-free condition followed by directed differentiation to macrophages with M-CSF either in serum-free media or in media with serum. Add the CFSE. succinimidyl ester (CFSE), a fluorescein derivative that partitions equally between daughter cells after cell division. Add an appropriate amount of complete M1- or M2-Macrophage Generation Medium DXF (C-28055, C-29056) to the cells, e.g. In this protocol, bone of L929-conditioned medium. Add 100 μL of the BMM/Nucleofector mix to the plasmid or siRNA. 36. Macrophage colony-stimulating factor (M-CSF) is a lineage-specific growth factor that is responsible for min. When isolated from blood and cultured in media with serum, adherent monocytes will differentiate into macrophages. Cells are isolated from bone marrow and cultured in vitro. Finally, the proliferation of the BMMs is assayed using carboxyfluorescein Add 1–2 μg of plasmid DNA or siRNA to a 1.5-mL tube. Hi. 5. Wash the BMMs twice with RPMI-1640. Although human M-CSF shows activity on mouse cells, mouse CSF shows no activity on human cells. Nice discussions on PBMC differentiation to macrophages by M-CSF . Incubate the cells in a humidified incubator with 5% CO2 at 37°C for 15 min. However, I intend to use frozen PBMCs for this purpose . (A) Morphological examination of cytospins using histological stains (e.g., May-Grünwald-Giemsa staining to visualize nuclei Sacrifice mice by cervical dislocation. Count the number of particles. MACS® GMP Recombinant Human M-CSF is designed for ex vivo cell culture processing. Tissue macrophages can be derived from monocytes. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for … BMMs are suitable for numerous applications including, but not limited to, the examples shown here.
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