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Fluorescence-activated cell sorting (FACS) and single clone selection

TH Tin-Yan Ha
SC See-Wing Chan
ZW Zhangting Wang
PL Patrick Wai Nok Law
KM Kai-Kei Miu
GL Gang Lu
WC Wai-Yee Chan
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hiPSCs were dispersed into a single-cell suspension by using ACCUTASE™. Cells were washed in PBS and were concentrated by centrifugation at 200 g for 3 min. PBS was discarded and the cell pellet was resuspended in 1 mL of BD stain buffer (BD, 554656). The cells were transferred to a flow tube with cell strainer cap to obtain single cell suspension. GFP-positive cells were sorted and collected by BD FACS Aria II Cell sorter. Sorted cells were plated on newly coated Matrigel 6-well plates supplemented with CloneR™ in mTeSR™1 medium with 1000, 2000, and 5000 cells seeded per well. mTeSR™1 medium supplemented with CloneR™ was changed daily. Single colonies were picked and transferred to newly-coated Matrigel 24-well plates. CloneR™ was lifted after single colonies were transferred to 24-well plates.

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