Oct 20, 2023
Cell Viability Assay (MTT Assay) Protocol
- 1Department of Biology, Misericordia University
Protocol Citation: aasirvatham 2023. Cell Viability Assay (MTT Assay) Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x3kdg1b/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: October 20, 2023
Last Modified: October 20, 2023
Protocol Integer ID: 89677
Abstract
The purpose of this experiment is to investigate the effects of forskolin-mediated cAMP activation on the viability of LPS-treated Schwann cells. The immortalized rat RT4-D6P2T (ATCC #CRL-2768) and S16 (ATCC #CRL-2941) cell lines were cultured and received one of the following treatments: 0.1, 1, or 10 μg/mL of LPS, in N2 media (control) or N2 media supplemented with 2 µM of forskolin, for 1, 3, 12, or 24 hours, and the CyQUANT MTT Cell Viability Assay Kit (Thermo Fisher) was used to perform the viability assay.
To perform one (1) MTT assay:
To perform one (1) MTT assay:
Aseptically culture immortalized rat RT4-D6P2T Schwann cells (ATCC, Cat #CRL-2768, Manassas, VA) or S16 Schwann cells (ATCC, Cat #CRL-2941, Manassas, VA) in Dulbecco’s Modified Eagle Medium (DMEM) (ATCC, Cat #30-2002, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher, Cat #16000044, Waltham, VA) and 1% penicillin/streptomycin (Pen-strep) (GIBCO, Cat #15140-015, Gaithersburg, MD)/amphotericin B (R&D Systems, Cat #B23192, Minneapolis, MN) at 37°C and 5% CO2 in poly-L-lysine (PLL)-coated dishes.
At 80% confluency, split and seed cells into DMEM (200 μL DMEM/well) in a PLL-coated 96-well plate at a density of ~35,000 cells/well.
Incubate cells in DMEM for 24 hours.
After 24 hours, aspirate the DMEM and wash each well 2-3x with 200 μL HBSS. After the last wash, add 200 μL N2 media (DMEM/F12, no phenol red [Thermo Fisher, Cat #21041025, Waltham, MA] supplemented with 5 μg/mL insulin [Sigma, Cat #91077C, St. Louis, MO] and 100 μg/mL apo-transferrin [Sigma, Cat #T1147, St. Louis, MO]) to each well.
Incubate cells in N2 media for 24 hours.
After 24 hours, prepare the forskolin-supplemented media by adding 5 μL of a 2 mM forskolin stock to 10 mL of N2 media.
Add 200 μL of the appropriate medium to each well following the plate layout.
After adding the media, add the appropriate LPS dose to each well following the plate layout. For a 0.1 μg/mL dose of LPS, add 2 μL of a 10 μg/mL LPS stock OR 20 μL of a 1 μg/mL LPS stock. For a 1 μg/mL dose of LPS, add 2 μL of a 100 μg/mL LPS stock OR 20 μL of a 10 μg/mL LPS stock. For a 10 μg/mL dose of LPS, add 2 μL of a 1 mg/mL LPS stock OR 20 μL of a 100 μg/mL LPS stock.
Allow cells to incubate in the different treatment combinations for the required incubation time (1, 3, 12, or 24 hours).
Prior to the end of the incubation period, prepare a 12-mM MTT stock solution by adding 1 mL of sterile PBS directly to the tube. Vortex to mix until the MTT powder has completely dissolved.
Wrap the vial of MTT solution in aluminum foil and store at 4°C until ready for use (good for up to 4 weeks).
After the incubation period, remove the 96-well plate from the incubator and warm the vial of MTT solution by rubbing in between hands for about 10 seconds.
Add 10 μL of MTT to each well, and incubate the plate for 2-3 hours.
At the hour-and-a-half mark, remove one bottle of SDS from the MTT assay kit and bring to room temperature by letting it sit for 20-30 minutes.
Once at room temperature, add 10 mL of a 0.01 M HCl solution to the bottle of SDS and slowly pipette the mixture to minimize bubble formation.
After the 2-hour incubation with MTT, remove the 96-well plate from the incubator and add 100 μL of the SDS/HCl mixture to each well.
Incubate the plate for an additional hour but continuously check for lysed cells with purple precipitate (could take up to 1.5-2 hours).
Once the cells have lysed completely, remove the plate from the incubator and read in the SpectraMax ABS Microplate Reader (Molecular Devices, San Jose, CA) at 570 nm.
Optical density is measured as an indicator of cell viability, with a higher optical density indicating more viable cells, and a lower optical density indicating less viable cells.