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