Mar 02, 2024
CellTitre Glo 2.0 Viability Assay
- 1Department of Biology, Misericordia University
Protocol Citation: aasirvatham 2024. CellTitre Glo 2.0 Viability Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3815l3p/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 01, 2024
Last Modified: March 02, 2024
Protocol Integer ID: 96036
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, 0.1, 1, or 10 μg/mL of LPS, in N2 media (control) or N2 media supplemented with 2 μM forskolin for 1, 3, 12, or 24 hours. The CellTitre-Glo 2.0 Viability Assay (Promega) was used to perform the viability assay.
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Plate Layout.jpg
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To perform one CellTitre-Glo Viability Assay:
To perform one CellTitre-Glo Viability 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 ~20,000 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 200μL N2 media (DMEM/F12, no phenol red [Thermo Fisher, Cat #21041025, Waltham, MA] supplemented with5 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.
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, remove the CellTitre-Glo 2.0 Reagent and allow to equilibrate to room temperature.
Allow cells to equilibrate to room temperature for approximately 30 minutes.
Add an equivalent volume of CellTitre-Glo 2.0 Reagent to the volume of cell culture medium in each well. Be sure this step is done in minimal light.
Mix the contents for 2 minutes to induce cell lysis.
Once the contents have been mixed, read the plate in the SpectraMax M4 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA)
Luminescence is measured as an indicator of cell viability, with a higher luminescence indicating more viable cells, and a lower luminescence indicating less viable cells.