Mar 28, 2025
Cell Viability Protocol using CellTiter-Glo 3D
- Marine Simonneau1
- 1LabTAU
Protocol Citation: Marine Simonneau 2025. Cell Viability Protocol using CellTiter-Glo 3D. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoo55xv4o/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: 125616
Abstract
This protocol provides a viability measurement for 3D culture.
Materials
ATP (Mw = 551.14 g/mol) (A7699-1G, Sigma-Aldrich)
spheroids
Incomplete DMEM/F-12 culture medium without FBS (1%L-Glutamine, 1% Penicillin/Streptomycin) (Gibco 21331-020)
CellTiter-Glo‱ 3D Cell Viability Assay (Promega G968A)
Aluminium
Shaker
Opaque 96-well plate for reading (Costar 96 Flat Bottom White Polystyrene)
Luminescence reader (Tecan Infinite M Plex)
Preparation of the CellTiter-Glo 3D Solution
Preparation of the CellTiter-Glo 3D Solution
●
Thaw the CellTiter-Glo reagent in the
fridge at 4°C the day before the experiment.
●
Before use, let the kit reach room
temperature for 30 minutes.
●
Gently
mix the solution.
Preparation of ATP Solutions at Different Concentrations for the Calibration Curve
Preparation of ATP Solutions at Different Concentrations for the Calibration Curve
Dilution of the ATP Solution
| ATP Concentration (µM) | 0 | 0.5 | 1 | 2 | 3 | |
| ATP at 10 µM (µL) | 0 | 10 | 20 | 40 | 60 | |
| DMEM without FBS (µL) | 200 | 190 | 180 | 160 | 140 |
●
Transfer the ATP solutions into the
opaque 96-well plate for luminescence
Preparation of Spheroids in the Opaque Plate
Preparation of Spheroids in the Opaque Plate
●
Transfer the spheroids from the
incubation plate to the opaque 96-well plate for reading.
●
Place the plate on the 28-magnet
plate (Plate Holding) and aspirate the medium.
●
Add 50 µL of DMEM without FBS per
well.
Addition of CellTiter-Glo and Incubation of the Opaque Plate
Addition of CellTiter-Glo and Incubation of the Opaque Plate
●
Add 50 µL of CellTiter-Glo per well
(into wells containing 50 µL of DMEM without FBS).
●
Cover the plate with aluminum foil to
protect the samples from light, as CellTiter-Glo is light-sensitive.
●
Incubate the plate at room temperature
for 25 minutes.
●
During this incubation period, shake
the plate on a vortex for 5 minutes at 2 rpm.
Luminescence Reading
Luminescence Reading
●
Set
up the device:
○
Shaking
mode: Orbital shaking
○
Shaking
time: 3 s
○
Attenuation:
None
○
Integration
time: 1000 ms
○
Settle
time: 0 ms
○
Temperature:
22°C
●
Place the plate without the lid on
the reader platform and position the plate.
●
Start the reading and save the Excel
file.