From 2007.igem.org

3- Flow Cytometry

Analysis of pathway-dependent Ste2 induction by flow cytometry was performed as follows: cultures were grown to early log phase (OD600=0.1-0.3) in complete synthetic dropout media and then treated with 1 μM α-factor (Zymo Research) to activate the pathway. At 60 min. aliquots of cultures were removed, treated with cycloheximide (5μg/mL), and dispensed into 96-well culture plates. Following incubation at room temperature for 1 hr in the dark to allow for GFP fluorophore maturation, plates containing treated cultures were analyzed with a BD LSR-II flow cytometer (BD Biosciences) using a high-throughput sampling module. 10,000 cells were counted for each reading, and GFP fluorescence was measured by exciting at 488 nm with a 100 mW Coherent Sapphire laser. Mean fluorescent values reported are the result of at least 3 independent samples.

3- Flow Cytometry

Analysis of pathway-dependent Ste2 endocytosis by fluorescence microscopy was performed as follows: cultures were grown to early log phase (OD600=0.1-0.3) in complete synthetic dropout media and then transferred to a Concavaline-A treated 96-well fluorescence plate. Individual wells were observed at 100x in a Nikon Eclipse T2000 microscope. Ste2 endocytosis was induced by addition of 1 μM α-factor (Zymo Research) to activate the pathway. Time-lapse experiments were done by taking pictures every 1.5 minutes, for a total of up to 2 hours.