TITLE: CellTitre Glo 2.0 Viability Assay
AUTHORS: aasirvatham

[DESCRIPTION]
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.

[STEPS]
SECTION: To perform one CellTitre-Glo Viability Assay:
1. 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.
SECTION: To perform one CellTitre-Glo Viability Assay:
2. 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.
SECTION: To perform one CellTitre-Glo Viability Assay:
3. Incubate cells in DMEM for 24 hours.
SECTION: To perform one CellTitre-Glo Viability Assay:
4. 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
SECTION: To perform one CellTitre-Glo Viability Assay:
5. Incubate cells in N2 media for 24 hours.
SECTION: To perform one CellTitre-Glo Viability Assay:
6. After 24 hours, prepare the forskolin-supplemented media by adding 5 μL of a 2 mM forskolin stock to 10
mL of N2 media.
SECTION: To perform one CellTitre-Glo Viability Assay:
7. 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. 
SECTION: To perform one CellTitre-Glo Viability Assay:
8. Allow cells to incubate in the different treatment combinations for the required incubation time (1, 3, 12, or 24 hours).
SECTION: To perform one CellTitre-Glo Viability Assay:
10. Allow cells to equilibrate to room temperature for approximately 30 minutes.
SECTION: To perform one CellTitre-Glo Viability Assay:
11. 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. 
SECTION: To perform one CellTitre-Glo Viability Assay:
12. Mix the contents for 2 minutes to induce cell lysis.
SECTION: To perform one CellTitre-Glo Viability Assay:
13. Once the contents have been mixed, read the plate in the SpectraMax M4 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA)
SECTION: To perform one CellTitre-Glo Viability Assay:
9. Prior to the end of the incubation period, remove the CellTitre-Glo 2.0 Reagent and allow to equilibrate to room temperature. 
SECTION: To perform one CellTitre-Glo Viability Assay:
14. 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.