For single-cell isolation, we recommend FACS isolation followed by culture in conditioned media.
FACS isolation of single cells
- Add 100 ul conditioned media (see below) to each well of a 96-well culture plate.
- Spin and resuspend cells to sort in PBS with 1% FBS.
- Sort 1 cell into each well of a 96-well plate (put 100 cells into well A1 - this helps to find the focal plane when looking for colonies).
- Seal the plates with parafilm and place them in a sealed box with damp tissue paper to avoid drying out. Place the sealed box in a fly cell culture incubator.
- Grow for 2-3 weeks and then check each well for colonies.
Making conditioned media
- Grow S2 or S2R+ cells to 100% confluency in a 10 cm or T75 flask (10% FBS media).
- Split cells 1:5 (2ml of cells and 8ml of fresh media) and culture for 3 days. Cells should be almost 100% confluent but without overlapping cells.
- Remove all media and replace with 10 ml fresh media. Immediately split cells 1:1.
- Grow for 16 hours (no longer). Check if the cells look healthy at this point.
- Extract and filter media to remove all cells.
- Dilute media 1:1 using fresh media with 20% FBS.
The conditioned media can be stored at 4 degrees C for a couple of weeks.