RAG01 is the patient cells, RAG02 are parent control cell lines
Assay
- RAG01 and RAG02 cells were thawed 7 days prior to seeding, for RAG01 medium was supplemented with a-tocopherol
- RAG01 and RAG02 cells are passaged 4 days prior to seeding, a-tocopherol was removed (important to remove a-toc 3 or 4 days prior)
- Bright field Imaging with IN Cell 2200 (GE Healthcare)
- Cell viability assay using CellTiter-Glo (Promega) with PHERAstar FSX (BMG Labtech)
Results
Contact [email protected] for CTG results for calibrating against reference compounds.
Observations
We did some assay validation studies and here are the properties for your reference. They were conducted in relation to a high throughput screen.
- The timing of compound/drug treatment on RAG01 is critical to exclude the artificial effect of a-Tocopherol in the original culture media. Otherwise the real effect may be masked by a-Tocopherol. You can use the suggested values above or do your own calibration by doing a pilot test to treat RAG01 with the highest concentration (1.25mg/mL) of the drug at different time points (4, 5, or 6 days after removal of a-Tocopherol) for 24 hours and then monitor the viability change of RAG01. Pick the time point(4, 5, or 6 days after removal of a-Tocopherol) that exhibits the highest viability-boosting effect (if there is any; if not, use a-Tocopherol instead of the study drug) and then use that condition for the protocol (PCR pretreatments)
- Strong increase in ATP was measured using CTG for RAG01 upon 72h treatment with ferrostatin-1 and α-tocopherol
- No increase in ATP is measured using CTG for RAG02 with any of the tested compounds
- CTG signal decreased over time, however is relatively stable up to 6 hours after addition of CTG
- All plates have an assay window >6 and Z’ ≥0.6, with very little baseline variability, which provides confidence in ability to accurately identify compounds positively affecting the growth rate of RAG01 cells
- CTG signal decreased over time, however is relatively stable up to 6 hours after addition of CTG
- We plan to read the screening plates between 10 min and 3 h after CTG addition