For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome

Hello. We're improving abcam.com and we'd welcome your feedback.

Hello. We're improving abcam.com and we'd welcome your feedback.

Infomation icon

We haven't added this to the BETA yet

New BETA website

New BETA website

Hello. We're improving abcam.com and we'd welcome your feedback.

Take a look at our BETA site and see what we’ve done so far.

Switch on our new BETA site

Now available

Search and browse selected products

  • A selection of primary antibodies

Purchase these through your usual distributor

In the coming months

  • Additional product types
  • Supporting content
  • Sign in to your account
  • Purchase online
United States
Your country/region is currently set to:

If incorrect, please enter your country/region into the box below, to view site information related to your country/region.

Call (888) 77-ABCAM (22226) or contact us
Need help? Contact us

  • My account
  • Sign out
Sign in or Register with us

Welcome

Sign in or

Don't have an account?

Register with us
My basket
Quick order
Abcam homepage

  • Research Products
    By product type
    Primary antibodies
    Secondary antibodies
    ELISA and Matched Antibody Pair Kits
    Cell and tissue imaging tools
    Cellular and biochemical assays
    Proteins and Peptides
    By product type
    Proteomics tools
    Agonists, activators, antagonists and inhibitors
    Cell lines and Lysates
    Multiplex miRNA assays
    Multiplex Assays
    By research area
    Cancer
    Cardiovascular
    Cell Biology
    Epigenetics
    Metabolism
    Developmental Biology
    By research area
    Immunology
    Microbiology
    Neuroscience
    Signal Transduction
    Stem Cells
  • Customized Products & Partnerships
    Customized Products & Partnerships

    Customized products and commercial partnerships to accelerate your diagnostic and therapeutic programs.

    Customized products

    Partner with us

  • Support
    Support hub

    Access advice and support for any research roadblock

    View support hub

    Protocols

    Your experiments laid out step by step

    View protocols

  • Events
    • Conference calendar
    • Cancer
    • Cardiovascular
    • Epigenetics & Nuclear signaling
    • Immunology
    • Neuroscience
    • Stem cells
    • Tradeshows
    • Scientific webinars
    Keep up to date with the latest events

    Full event breakdown with abstracts, speakers, registration and more

    View global event calendar

  • Pathways
    Cell signalling pathways

    View all pathways

    View all interactive pathways

Mouse embryonic stem cells to macrophage-like cells

Related

  • Fluorescent staining of viable cells
    • Guidelines for small molecule inhibitors
      • Hepatocyte differentiation protocol

        A robust 20 day differentiation protocol to direct mouse embryonic stem cells (mESCs) towards the macrophage lineage.

        Print this protocol.

        View our macrophage differentiation protocol summary.

        Macrophages display plasticity in both phenotype and function; therefore standardized culture and differentiation protocols are required to ensure consistency between different cell populations. Throughout this protocol we refer to these cells as embryonic stem cell-derived macrophages (ESDMs).

        Edited protocol reproduced with kind permission of Lesley Forrester and the Journal of Immunological Methods.

        Preparation of media for differentiation of mESCs to ESDMs

        All media preparation should be carried out in a tissue culture hood under aseptic conditions.

        1. Preparation of L929 conditioned medium*
          1. For L929 medium, supplement DMEM/F12 GlutaMAX™ with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin.
          2. Swirl to mix components and store at 4°C.
          3. Harvest conditioned medium (CM) containing colony stimulating factor (CSF-1) from adherent L929 cells 3 days following confluency. Filter through a 0.22 µm membrane to remove cell debris and store at -20°C.
        2. Preparation of GMEMSR​ medium for mESC maintenance
          1. ​For GMEM​​SR ​medium, supplement GMEM with 10% (v/v) FBS, 1% (v/v) non-essential amino acids (of a 100X solution), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol and 100 U/mL leukemia inhibitory factor (LIF).
          2. Swirl to mix components and store at 4°C.
        3. Preparation of ESDMDiff​ medium for macrophage differentiation
          1. ​For ESDM​Diff medium, supplement GMEM with 10% (v/v) FBS (pre-screened for optimal hematopoietic differentiation**), 1% (v/v) non-essential amino acids (of a 100X solution), 2 mM L-glutamine, 1 mM sodium pyruvite, 0.1 mM 2-mercaptoethanol, 1 ng/mL IL-3 and 15% (v/v) L929 CM (contains CSF-1).
          2. Swirl to mix components and store at 4°C.
        4. Preparation of ESDM​Cult medium for macrophage maturation and growth 
          1. ​​​For ESDMCult​ medium, supplement GMEM with 10% (v/v) FBA (pre-screened for optimal hematopoietic differentiation**), 1% (v/v) non-essential amino acids (of a 100X solution), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 15% (v/v) L929 cM (contains CSF-1), 100 U/mL penicillin and 100 µg/mL streptomycin.
          2. Swirl to mix components and store at 4°C.


        * L929 conditioned medium is an alternative source of macrophage colony stimulating factor (CSF-1). L929 cells (ATCC) are derived from mouse subcutaneous tissue.

        ** FBS was pre-screened for optimal hematopoietic differentiation using methylcellulose assay for the detection of hematopoietic progenitors.



        Generation of ESDMs

        1. Culture mESCs (E14) on 0.1% gelatin-coated tissue culture flasks in GMEMSR​ medium.
        2. Initiate macrophage differentiation by seeding 6x10​5 trypsin-dissociated mESCs in suspension culture in 20 mL ESDMDiff per 95 mm bacteriological-grade Petri dish (Day 0). During the first 4 days the mESCs should start to differentiate and form embryoid bodies (EBs).
        3. On Day 4, to avoid adherence the EBs should be gently dislodged and transferred to new Petri dishes in fresh ESDM​Diff medium using a wide-bore pipette.
        4. Repeat Step 3 again on Day 6, to ensure that the EBs do not attach to the Petri dishes. 
        5. On Day 8 transfer the EBs to 95 mm gelatin-coated tissue culture dishes, in fresh ESDM​Diff medium. The EBs will become adherent after 2 days.
        6. At Day 10 collect the medium that contains the non-adherent macrophage precursor cells, centrifuge, re-suspend in 20 mL ESDM​Cult and plate on 95 mm diameter bacteriological Petri dish. At the same time add 20 mL of fresh ESDMDiff medium to the original plate of adherent EBs, so that non-adherent cells can then be harvested again 2 days later. Harvesting in this way can be repeated every 2 days for up to a total of 20 days.
        7. Between Day 12 and 17 of this differentiation, ESDMs will adhere to the plastic Petri dishes and proliferate to form a confluent monolayer. For functional experiments that are performed in suspension, cells can be cultured for 1 day prior to experiments in Teflon® pots. Between Days 15 and 20, ESDMs can be harvested for testing. To do this, wash twice in PBS, then add 5 mL TVP (1 mM EDTA, 1% chicken serum, 0.025% Trypsin) solution and incubate for 20 min at 37°C. Add 10 mL serum-containing medium to stop the trypsin, centrifuge, and wash once with PBS before use in further experiments.


        Since each dish of mESCs can yield 2–4x10​6 ESDMs and supernatant can be collected 6 times (i.e. every 2 days from Day 10–20), a total of 12–24x10​6 ESDMs can be harvested from one starting dish of 6x10​5 mESCs.



        Taken from:

        Zhuang L, Pound JD, Willems JJ, Taylor AH, Forrester LM and Gregory CD (2012). Pure populations of murine macrophages from cultured embryonic stem cells. Application to studies of chemotaxis and apoptotic cell clearance. J Immunol Methods, 385, 1-2.

        Discover more
        Get resources and offers direct to your inbox Sign up
        A-Z by research area
        • Cancer
        • Cardiovascular
        • Cell biology
        • Developmental biology
        • Epigenetics & Nuclear signaling
        • Immunology
        • Metabolism
        • Microbiology
        • Neuroscience
        • Signal transduction
        • Stem cells
        A-Z by product type
        • Primary antibodies
        • Secondary antibodies
        • Biochemicals
        • Isotype controls
        • Flow cytometry multi-color selector
        • Kits
        • Loading controls
        • Lysates
        • Peptides
        • Proteins
        • Slides
        • Tags and cell markers
        • Tools & Reagents
        Help & support
        • Support
        • Make an Inquiry
        • Protocols & troubleshooting
        • Placing an order
        • RabMAb products
        • Biochemical product FAQs
        • Training
        • Browse by Target
        Company
        • Corporate site
        • Investor relations
        • Company news
        • Careers
        • About us
        • Blog
        Events
        • Tradeshows
        • Conferences
        International websites
        • abcam.cn
        • abcam.co.jp

        Join with us

        • LinkedIn
        • facebook
        • Twitter
        • YouTube
        • Terms of sale
        • Website terms of use
        • Cookie policy
        • Privacy policy
        • Legal
        • Modern slavery statement
        © 1998-2023 Abcam plc. All rights reserved.