Hello
We did an experiment with equine chondrocytes from the metacarpophalangeal joint. We incubated them for 2 days on a 96-well plate and then we wanted to detach them with trypsin/EDTA 0.05 %. The problem was that they did not detach even after 1 hour (we checked every 5 minut). Therefore we tried with trypsin/EDTA 0.25 % and same thing happend the cells did not detach. Therefore we tried to incubate for 5 days, but still the results were the same.
Have anyone experienced something similar?
We are considering trying accutase instead of trypsin. Does anyone have experince with accutase or maybe something else?
Best Regards
Jeppe and Christoffer
Did you wash each well with PBS before adding the trypsin ? Leftovers from the medium will stop the enzyme from functioning.
Hello Maximilian
We did not wash with PBS, because we removed our culturemedium and then stimulated our cells with local anesthetics. We then removed the anesthetics and then we added trypsin.
Do you know if the anesthetics will also stop the enzyme from functionin?
Thanks a lot for your answer
The function of Trypsin is dependent on the pH. The optimal range seems to be 7-8. Which anesthetic did you use ?
We used mepivacain, bupivacain, lidocain, xylocain and ropivacain all with the same result
Might washing with PBS after the stimulation be a possibility ?
Yes we will try to do that
thanks a lot for your help Maximilian :)
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating