Approximately 200 E(spl)m4-GFP wing imaginal discs were dissected from late third-instar larvae and collected in ice-cold PBS. Discs were transferred to a 5-ml Falcon tube containing 1 ml of 9X trypsin-EDTA (Sigma)/1X PBS and dissociated at room temperature with gentle rocking for 2 hours and vigorous pipetting every 15 minutes for another hour. Before sorting, the viability of the dissociated cells was confirmed by examining a small trypan blue-stained sample in a hemacytometer. The background fluorescence of wing imaginal disc cells was determined by analyzing w1118 disc cells using FACS. All E(spl)m4-GFP wing disc cells with fluorescence greater than that of w1118 control cells (GFP-positive cells) and 100,000 cells with fluorescence below the w1118 control threshold (GFP-negative cells) were sorted directly into 600 μl of RLT lysis buffer (Qiagen). Keywords = Proneural Keywords = PNS Keywords = PNC Keywords = SOP Lot batch = 2004163
