Protocol:

1. Seed (number, cell type) cells per well in a 96-well plate in (volume) μL of (type) media.
2. Incubate the plate for 48 hours under experimental conditions.
3. Add 10 μL of the CCK-8 reagent (not sponsored; we use it – Abcam, cat. No. 228554) to each well.

Pro Tip: Prepare a mix of CCK-8 (10 μL) and serum-free media (100 μL) for all wells, and add 110 μL of mix into each well. Use the multi-channel pipette to load the mix into wells.

5. Incubate the plate for an additional 1-2 hours.
6. Measure the absorbance at 460 nm with a 540 nm reference using a spectrophotometer. The water-soluble tetrazolium salt WST-8 5-(2,4-disulfophenyl)-3-(2-methoxy-4-
   nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazolium, inner salt, monosodium salt is taken up and reduced in the mitochondria of viable cells to a soluble orange formazan dye. The absorbance is directly proportional to the number of viable cells.
7. Normalize the cellular proliferation to the experimental control.

Key Notes:

(a) To save time, use the pro tip.

(b) Calibrate the number of cells, so there is minimal variation between duplicates and triplicates of each sample.

(c) Calibrate the number of cells so the supernatant can be collected at the same time for ELISA analysis. 

(d) The method does not require cell lysis, so it is possible to move on to additional cell-based assays or downstream applications without killing cells [1].

To download the template, use the embedded document:

Bibliography:

1. JEAN-FRANÇOIS TÊTU. Do you use MTT or WST-1 to count cells? Created on 20-05-2016. Accessed on the Internet [14 Feb, 2023] via the link: https://www.tebu-bio.com/blog/still-using-mtt-or-swt-1-for-cell-counting/