2 Materials
Prepare sorting/staining buffers prior to beginning and store in
sterile conditions at 4
C. This can be stored for several months.
Add fresh DNAse to an aliquot just prior to use (see Note 1).
2.1 Sorting/Staining
Buffers
1. FACS Buffer.
(a) Add the following ingredients to 1 L HBSS without Ca
2+
and Mg
2+
: 25 mL HEPES (stock 1 M), 5 mL MgCl 2
(Stock 1 M) (see Note 2), 10 mL Pen/Strep, 5 mL
Amphotericin B, 10 ml NEAA (stock 100Â), 10 mL Lglutamine (Stock 100Â), 3 g Glucose, 10 mL Sodium
Pyruvate (Stock 5% FBS).
B
Pre - FACS sample
Enriched EMT cells
Enriched epithelial cells
Combined histogram
E-cadherin
E-cadherin
E-cadherin
E-cadherin
A
Fig. 1 (a) Overall strategy for isolating epithelial (ECAD+/YFP+) and mesenchymal (ECAD-/YFP+) tumor cells
by FACS. (b) Purity of epithelial and mesenchymal populations after FACS. Left (Red): tumor cells before
applying to a MACS column. Left-center (purple) enriched ECAD+ epithelial cells after LS column. Right-center
(blue): enriched ECAD- mesenchymal cells after LD column. Right: merge of the three histograms. Upper
corners demonstrate percent of purity of E-cadherin
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