Single-cell Cloning and Expansion

Bulk multifluorescent cell lines were single cell-flow sorted using a BD FacsAria^TM III instrument (BD Bioscience) where single cells were sorted unbiased by any marker expression directly into the inner 60 wells of 5 laminin (Millipore) pre-coated flat-bottom 96-well plates (PerkinElmer). Single cells were dropped in 100 μL/well of the same medium as described above. The outer 16 wells were filled in with 200 μL/well of PBS to avoid evaporation of medium. After single cell-flow sorting, the plates were incubated at 37 °C, 5% CO₂, and colonies monitored as previously described [12 (link)]. Once weekly cells were refed with 25 μL of medium/well and plates scanned on a CeligoS cytometer (Nexcelom Bioscience, Lawrence, MA, USA) using the Confluence application, to evaluate colony growth. Single cell-derived colonies were detached using Accutase (Euroclone, Pero, Italy) when they achieved approximately 60–80% confluency and collected for further expansion to stably derive single cell-derived clones.