Mar 28, 2025
Cell viability using Calcein
- magali perier1
- 1INSERM U1032
Protocol Citation: magali perier 2025. Cell viability using Calcein. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoo5yxv4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 28, 2025
Last Modified: March 28, 2025
Protocol Integer ID: 125640
Abstract
This protocol measures the viability of cells within a spheroid after dissociation.
Materials
Spheroids
DPBS
Accumax (SCR006 sigma-aldrich)
24-well plate cell-repellent
for culture (Greiner Bio-one 662970)
Calcein AM-blue (c1429 Invitrogen)
agitator incubator
(Incu-shaker)
Flow cytometer (Fortessa)
Spheroid dissociation
Spheroid dissociation
-Place spheroids in a 24-well cell repellent plate, pooling 8
spheroids in a well.
-Remove medium and add 500µl/well of DPBS
Remove
DPBS and add 500µl/well of accumax
Flush
to break up spheroids
Incubate
plate at 37°C in incu-shaker at 200rpm for 1h30, flushing regularly (45' +45'
for hearts/crowns).
Recover and place in 2ml tubes
Calcein AM Blue staining
Calcein AM Blue staining
- Centrifuge 300g 5min - discard supernatant
- Add 500µl /tube of DPBS - suspend cells -
centrifuge again - discard supernatant
-
Add 500µl /tube of Calcein 7µg/ml
- Incubate for 15 min in the dark
Centrifuge - add 300µL/tube of DPBS
- Run on flow cytometer