S1400exi flow cytometer

Choroidal cells were isolated as described above and fixed in 4% formaldehyde at 4°C overnight. Cells were then rinsed in wash buffer (PBS + 2% BSA), following by incubation in blocking buffer (2% BSA, 0.2% Triton X-100 in PBS) for 10 min at room temperature. Cells were immunolabelled with anti-chick CHRNA3 (1:4000) in blocking buffer overnight 4°C with rotation. Cells were then washed 3 × 10 min with wash buffer followed by incubation in goat anti-rat alexafluor 647, diluted 1:1000 in wash buffer for 1 hr at room temp with rotation. Cells were then washed 3 X 10 minutes with wash buffer, nuclei were labelled with DAPI (1:1000 in wash buffer), and stored in wash buffer at 4°C in the dark prior to FACS analysis. Stained cells were subjected to data acquisition using a Stratedigm S1400Exi flow cytometer (Stratedigm, Inc., San Jose, CA) using an excitation of 640 nm and the emission collected using a 676/29 nm band pass filter. Data were analyzed by FlowJo software.