cbil.upenn.edu:RAD.Protocol:5577:Protocol - cbil.upenn.edu:RAD.Protocol:5577:Protocol
Dissected pancreases or duodenums from E15.5 ngn3EYFP/+ embryos were incubated in a solution of 0.05% trypsine-EDTA (GIBCO) at 37 degrees C for 5min. The digestion was stopped by adding F10 with 10% (vol/vol) FCS. The resulting suspension of single cells was washed twice with PBS and filtered through a 70um nylon-mesh. Flow cytometric analysis and sorting of EYFP-labelled progenitors were carried out using a FACS Vantage SE DiVa high speed cell sorter with DiVa software (Becton Dickinson Biosciences, San Jose, Ca.). EYFP expressing cells were detected after excitation with the 488 nm line of an Argon Ion Laser (200 mW) using the signal area of the FL 1 (530/30 nm BP) and FL 2 (585/42 nm BP) channels, separated by a 560 SP dichroic filter (corresponding to the standard FITC and PE channels of the sorter). Sorting of the EYFP positive cells was performed using a 100 um flow cell at 40 psi and 52kHz.