Anti cd39 magnetic beads

Cx3cr1^gfp/+ iPSC were differentiated into microglia using a two-stage protocol. In the first stage, a modified protocol for the hematopoietic differentiation of iPSC^{40 (link)}, Cx3cr1^gfp/+ iPSC colonies were dissociated into a single-cell suspension using 1 mg/mL collagenase (Invitrogen) and Accutase (Stemcell Technologies) and plated at a density of 1.5e6 cells per 15-cm tissue culture dish of confluent OP9 stromal cells. This Stage 1 culture was maintained in alpha-MEM with 10% defined FBS (Hyclone), 2-mercaptoethanol, and penicillin/streptomycin for seven days. On day four, the media was fully replaced. After seven days of Stage 1 culture, cells were harvested by dissociation with trypsin (Gibco) and transferred onto a confluent astrocyte monolayer for Stage 2 culture. Stage 2 cultures were maintained in DMEM/F12 media (Gibco) with 10% defined FBS, 2 mM 2-mercaptoethanol, 2 mM Glutamax (Gibco), non-essential amino acids (Gibco), 20 ng/mL recombinant murine GM-CSF (Peprotech), 20 ng/mL recombinant murine M-CSF (Peprotech), 20 ng/mL recombinant murine IL-3 (Peprotech), and penicillin/streptomycin. Media in Stage 2 cultures was replaced twice weekly. Purification of iPS-MG from Stage 2 cultures was carried out using anti-CD11b or anti-CD39 magnetic beads (Miltenyi) according to manufacturer’s instructions followed by optional FACS sorting of iPS-MG by gating on GFP⁺ cells.