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. 2009 Jan 12;106(5):1380–1385. doi: 10.1073/pnas.0812291106

Fig. 4.

Fig. 4.

Flow cytometry selection. (A and B) Flow cytometry plots from selection rounds 3 and 4 respectively, with the sort gate for selection indicated in blue. (C and D) Both from round 5 but use different fluorescence markers. (A–C) Anti-c-myc-Alexa Fluor 647 was used for scFv visualization and anti-phage/Zenon-PE for phage binding (the phage-negative populations appear different because they were obtained on two separate instruments with different voltage settings). (D) Anti-c-myc-Alexa Fluor 488 was used for scFv visualization and anti-HA-Alexa Fluor 647 for phage binding. (E) Input and output titers for both phage and yeast for each selection round and the percentage of phage and yeast that are positive for the desired Z13e1–TJ1D antibody–antigen pair. The percentage of positive phage was determined by phage ELISA for 48 clones after each round. The percentage of positive yeast cells was calculated by determining the percentage of scFv-positive yeast cells that bound to phage during the flow cytometry selection; this number actually reflects the percentage of double positive yeast cells that are input into the selection round, not the output from that round.