Anti cd117 beads

Picovitro slides (Picovitro) were first coated with Collagen type I, followed by culture of OP9 stromal cells, previously irradiated with 30 Gy, at a number of 10² per well in dendritic cell conditioned medium [4 (link)]. Cultures were left overnight for OP9 adherence. The following day, HSCs were prepared from c-kit (CD117)-enriched BM from either C57BL/6, UBC-GFP mice using anti-CD117 beads (Miltenyi), followed by staining with antibodies against Sca-1, ckit and IL-7R. Progenitors were sorted as Sca-1⁺c-kit^hiIL7R-progenitors [5 (link)]. PDMS membranes were layered on top, and slides fixed to a custom-bracket to fully encapsulate slides in order to prevent dehydration but allow gas exchange. The slide and bracket were mounted in a Zeiss Live Cell Observer, with 5% CO²-in-air, and at 37° C. Phase contrast images of multiple positions in the slide were taken with a 10x Plan-Neofluar phase contrast objective with a 1.6x Optovar every 2 minutes. GFP images were captured every hour, with each clone being GFP+ or GFP- throughout the image sequence. The GFP information was used for a separate validation experiment, although it was helpful during validation here for resolving tracking ambiguities.