For single-cell isolation, we recommend FACS isolation followed by culture in conditioned media.
Making conditioned media
- Grow several T75 flaks of S2 or S2R+ cells in log phase growth with regular media to 60-70% confluency (~2 days after splitting 1:10).
- Remove all media from flasks without disturbing the cells and filter through a 0.2 µm filter.
- Dilute 1:1 with regular media and filter again through a fresh 0.2 µm filter.
- The conditioned media can be stored at 4 degrees C for a couple of weeks. Quality varies batch by batch.
FACS isolation of single cells
Add 100 µL conditioned media to each well of a 96-well culture plate.
- Plates will sit untouched for a several days. We have found that creating a mini-humidity chamber in the plate helps with cell growth. To do this, add 200 µL regular media to the border wells and 100 µL of conditioned media to all internal wells.
- Resuspend cells in media and count on hemacytometer. Less than 1x10^7 cells are needed for FACS.
- Filter the appropriate amount of cells through a 40 µm filter.
- Spin and resuspend cells to sort in 1x PBS with 1% FBS.
Sort 1 cell into each internal well of a 96-well plate
- Put 100 cells into well B2 - this helps to find the focal plane when looking for colonies).
- Seal the plates with parafilm and incubate at 25ºC and 33% RH for ~20 days.
- Check each well for colonies.
How to maintain single cell clones in culture
After 20 days, large and healthy clones should be visible by eye as small white specks. Marks these wells and check under the microscope, as well as all other wells for colonies that didn’t grow big enough to be seen by eye.
- Small colonies can still survive and be expanded if not many large colonies formed; however, they will require extra care during the first few expansions.
Add 50 µL of fresh regular media to the wells chosen for expansion and resuspend the cells to disperse them across the well and encourage growth.
- Alternatively, after resuspending cells, the full volume can be transferred to the well of a new 96-well plate. The benefit to this is that it is out of an unhealthy environment that may be contaminated due to old media. The downside is that come cells might be lost, which would be particularly detrimental for smaller colonies. If you are certain of contamination, transfer the cells.
- Check daily. When confluent, expand cell line as previously described.