Mar 28, 2025
Cell viability using PrestoBlue HS
- Jacqueline Ngo-Reymond1
- 1INSERM U1032
Protocol Citation: Jacqueline Ngo-Reymond 2025. Cell viability using PrestoBlue HS. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4z7k2vo5/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: 125641
Abstract
This protocol measures the viability of cells cultured in 2D.
Materials
KPC A219
iMEF
DMEM/F-12 culture medium (Gibco 21331-020) with FBS , 1%
L-Glutamine and 1% Penicillin/Streptomycin
DPBS
Trypsin EDTA 0,05%
96-well plate for culture bottom F
PrestoBlue HS Cell Viability Reagent (Invitrogen P50200)
Aluminium
Opaque 96-well plate for reading (Perkin Elmer Optiplate
96-F blake Polystyrene 6005270)
Fluorescence reader (Horiba Fluoromax-4)
1. Day one : Cell seeding
1. Day one : Cell seeding
Cells should
not be confluent the day of test. They should be in exponentiel phase of growth.
At confluency, cells may lower their metabolic rate. So before experiment, the
appropriate cell density should be determined.
- Warm the complete DMEM/F12 medium at 37°C
- Under biosafety hood, aspirate the medium in 75cm² flask
-Add 5 ml of DPBS to rinse the cell and aspirate the PBS
- Add 2 ml of trypsin EDTA 0,05% and incubate at 37°C for 5 minutes. Verify that cells were detached from the flask wall. Add 10 ml of medium and flush 3 times to separate cells
-Take 20 µl of cells of each cell line and count
-Prepare cell suspension to have 4000 cells in 100 µl for drug incubation for 24 hours
and 2000 cells in 100µl for 72 hours
- Dispense 100 µl in each well of 96-well plate
- Place the 96-well plates in incubator at 37°C, 5% CO2
2. Day 2 : Drug treatment
2. Day 2 : Drug treatment
- prepare solution to have 0-50-100-200 nM of Gemcitabine
- Aspirate the medium in 96-well plate
- Dispense 100µl of differents solution
-Incubate for 24 to 72 hours at 37°C, 5% CO2
3. Day 3-5 : PrestoBlue Test
3. Day 3-5 : PrestoBlue Test
Warm PrestoBlue HS Cell Viability Reagent to room temperature
Aspirate the medium in each well
Add 90 µl of fresh medium in each well
Add 10 µl per well of PrestoBlue HS Cell Viability Reagent
Place the 96-well plate in incubator at 37°C, 5% CO2 for 1 hour
Transfer medium of each well in a opaque black 96-well plate
Record fluorescence on plate reader (Enter value of emission (560nm) and excitation (590nm) wavelenght)