XTT Assay

1. Prepare cells in a 96-well plate (100 µL/well).
2. Add 50 µL of XTT Solution to each well.
3. Incubate at 37°C for 1 - 4 hours.
4. Monitor absorbance at OD = 450 nm.

For guidelines on cell sample preparation, please visit:
https://www.aatbio.com/resources/guides/cell-samplepreparation.html

1. Plate 5000 to 10,000 cells/well in a tissue culture microplate with clear bottom.
2. Add test compounds into the cells and incubate for a desired period of time (such as 24, 48, or 96 hours) in a 37°C, 5% CO2 incubator. For blank wells (medium without the cells), add the same amount of test compounds. The suggested total volume is 100 µL for a 96-well plate or 50 µL for a 384-well plate.
   Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for proliferation or cytotoxicity induction. For proliferation assays, use fewer cells; for cytotoxicity assays, use more cells to start with.
3. Add 50 µL/well (96-well plate) or 25 µL/well (384-well plate) of XTT solution to each well.
4. Incubate the plate at 37°C for 2-5 hours, protect from light.
   Note: The incubation time could be from 30 minutes to overnight depending on the individual cell type and cell concentration used. Optimize the incubation time for each experiment
   
5. Monitor the absorbance increase with an absorbance plate reader at OD = 450 nm and 660 nm.
   Note: The 660 nm absorbance reading is used to eliminate the background signal contributed by cell debris or other non-specific absorbance.
6. Determine the specific absorbance of the sample using following formula:
   [Abs450 nm(Test) – Abs450 nm(Blank)] – Abs660 nm(Test).

1. Prepare cell culture in a tissue culture microplate with a clear bottom. The suggested total volume is 100 µL for a 96-well plate or 50 µL for a 384-well plate.
   Note: We used serially diluted HeLa and Jurkat cell suspension in a clear bottom 96-well plate for the assay.
2. Add 50 µL/well (96-well plate) or 25 µL/well (384-well plate) of XTT Solution to each well.
3. Incubate the plate at 37°C for 2-5 hours, protect from light.
   Note: The incubation time could be from 30 minutes to overnight depending on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
4. Monitor the absorbance increase with an absorbance plate reader at OD = 450 nm and 660 nm.
   Note: The 660 nm absorbance reading is used to eliminate the background signal contributed by cell debris or other non-specific absorbance.
5. Determine the specific absorbance of the sample using following formula:
   Specific Absorbance = [Abs450 nm(Test) – Abs450 nm(Blank)] – Abs660 nm(Test).