Dear Jorrit,

I used accutase + gentle mechanical dissociation with fire-polished pasteur pipette (coated with FBS). It was working quite well

All the best

How big are your spheroids?

Usually I trypsinize spheroids of diameter 150-300 um. I wash them several times with PBS. Then incubate spheroids in TrypleExpress for 15 min. at 37C (15 ul into one well of 96-well plate). Then add 85 ul of medium and pipette 10-20 times (depending on cell line I use).

I used accutase after washing the spheroids with 1XPBS. Accutase is not working at 37C. Just to leave in room temperature. Did you do this?

Dear Flavie, Vilma and Jamail,

Thanks you for your suggestions.

Flavie, why do you need the FBS coating?

Vilma, the spheroids are 400-600µm in size, depending on the day of harvesting. You first use trypsin and then TrypLE Express?

Jamail, indeed, I have used the accutase at 37°C. I did not know this, as it works for dissociating my monolayers at 37°C... You did not combine it with a mechanical dissociation step?

Best

FBS is to avoid the sticking of the cells on the glass.

If you have a lot of cells you can also use a strainer 40µm at the end of dissociation.

No need to use other way of mechanical dissociation at all except re-suspended gently with micro-pipette. It is absolutely not recommended to use Accutase at 37C.

Another one to be careful is that you should not keep sphere size upto 400 to 600 um. After you have done dissociation by whatever method, you have to use cell strainers before preparation for flow. You can have 20 um cell restrainers.

Dear Dr. Ming Chuan Yu,

Why it's not recommended to use Accutase at 37C ? It can affected the cell viability ? Do you have recommendations for the optimal use of Accutase to dissociate spheroids ?

Thanks and best,

Benjamin