Dataset Open Access
Alasoo, Kaur; Rodrigues, Julia; Gaffney, Daniel
Human induced pluripotent cells (iPSCs) were obtained from the HipSci project (http://www.hipsci.org) and differentiated into macrophages using an established protocol (van Wilgenburg, 2013). The genotype_id column of the flow_sample_metadata.txt file contains the canonical HipSci iPSC line name from which the macrophages were differentiated.
We used flow cytometry to measure the cell surface expression of three canonical macrophage markers: CD14, CD16 (FCGR3A/FCGR3B) and CD206 (MRC1). Macrophages were cultured in 10 cm tissue-culture treated plates and detached from the plates by incubation in 6 mg/ml lidocaine-PBS solution (Sigma L5647) for 30 minutes followed by gentle scraping. From each cell line we harvested between 300,000-500,000 cells. Detached cells were washed in media, centrifuged at 1200 rpm for 5 minutes and resuspended in flow cytometry buffer (2% BSA, 0.001% EDTA in D-PBS) and split into two wells of a 96-well plate. Nonspecific antibody binding sites were blocked by incubating cells with Human TruStain FcX (Biolegend) for 45 minutes and washing with flow cytometry buffer. Half of the cells were stained for 1 hour with the PE-isotype control (BD 555749) antibody. The other half of the cells were co-stained for 1 hour with following three antibodies: CD14-Pacific Blue (BD 558121), CD16-PE (BD 555407), CD206-APC (BD 550889). After staining, the cells were washed three times. Resuspended cells were filtered through cell-strainer cap tubes (BD 352235) and measured on the BD LSRFortessa Cell Analyzer.