TABLE 2 Troubleshooting table
Step
Problem
Solution
Add an increased number of FGSCs from Step 11 to the
plate or use 48 or 96 well plates to increase FGSC
density.
The confluence of the mitotically inactivated STO cell
monolayer should be 90%.
Various cell culture procedures including medium
changing and subculture should be performed gently.
14
FGSCs proliferate
very slowly
Also, centrifugation time must be 5 min for pelleting cells
during the subculture procedure.
Do not split FGSC cultures during the first week of
culture.
Collect the supernatant before changing the medium
during the first 2–3 weeks, and centrifuge the supernatant
at 15294 g for 5 min at 4 °C. Then, filter through a 0.22
µm pore-size filter. This medium (100–200 µl per well)
can be added to FGSC cultures.
Long-term culture of FGSCs should use FGSC medium.
20
Long-term culture
of FGSCs fail
Digestion time
optimized.
for FGSC
subculture
should be
Achieve a single cell suspension by pipetting FGSC
clusters during subculture.
Avoid contamination of FGSC cultures.