TITLE: EPCAM Sorting Protocol March
AUTHORS: Morrisey Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Epcam sorting Protocol</span></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-weight:bold;">FACS Buffer: </span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">- 2mL EDTA (Invitrogen, cat# 15575020, UltraPure, 0.5M, ph 8.0, 100mL)</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">Countess™ Cell Counting Chamber Slides (Thermo Fisher cat#C10228)</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">ViaStain™ AOPI Staining Solution (Nexcelom/ Fisher cat #CS2-0106)</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">Evos (Lifetech)</div><style>
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[STEPS]
?. Make sure 4C centrifuge is on and at correct temperature.
?. After completing HT2-280 sorting protocol centrifuge HT2-280 – cells for 5 min @ 300g(rcf).
?. Resuspend pellet in 100uls αEpcam microbeads and 900uls FACS.
?. Incubate in 4C for 30 min in darkness.
?. Wash pellet with 20mL FACS and spin for 5 min @ 300g(rcf).
?. Resuspend pellet in FACS (the # of ml should = 2x the number of columns being used).
?. Set up columns and wash with 3mL FACS 3x.
?. Add 2mL of cells per column.
?. Wash each column 3x with 3mL FACS.
?. 10.  Discard flow through-- negative cells.
?. 11.  Plunge all columns into 1 tube using 5mL FACS per column.
?. 12.  Spin +cells for 5 min @300g(rcf) and resuspend in 0.04%BSA+PBS.
?. 13.  Count cells and record viability on cellometer (ViaStain) or EVOS using trypan blue (1:1).
?. 14.  Proceed to cytospin protocol, organoid protocol, or bank cells for RNA and/or protein.