Cell Counting Kit-8

Description

Catalog No.: CSK1160
Kit Content
CCK-8 solution 5ml
Storage
Store at 4℃ in dark for one
year
Introduction
Cell Counting Kit-8 (CCK-8) allows very convenient assays by
utilizing Dojindo’s highly water-soluble tetrazolium salt. WST-8 [2-
(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-
(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt]* produces a
water-soluble formazan Dye upon reduction in the presence of an
electron carrier,as shown in Figure 1.Cell Counting Kit-8 is a onebottle solution; no premixing of components is required. Cell
Counting Kit-8, being nonradioactive, allow sensitive colorimetric
assays for the determination of the number of viable cells n cell
proliferation and cytotoxicity assays. WST-8 is educed by
dehydrogenases in cells to give a yellowcolored roduct (formazan),
which is soluble in the tissue culture medium. The amount of the
formazan dye generated by the activity of dehydrogenases in cells
is directly proportional to the number of living cells. The detection
sensitivity of CCK-8 is higher than other tetrazolium salts such as
MTT, XTT, MTS or WST-1.
The kit components are sufficient for performing up to 500
assays.

Protocol
1. Collect logarithmic phase cell, adjust cell suspension concentration; add 100ul floor plate. In general,
cells seeded at densities between 1000-10,000 cells per well (side holes filled with aseptic PBS
buffer).
2. Seed cells in a 5% Co2 incubator at 37℃ until cells bespread well bottom for one floor (cells number
for each well is according to cells’ size and breed speed). Add concentration gradient drug.
Principlely, add drug after cells adhere. 0-10ul per well. Using 3-5 repeating pipettors.
3. Add 10μl CCK-8 into each well.( considering the ratio 1:10). Choose the wells without cells as
contrast wells.
4. Incubate for 0.5-4 hours, usually 1 hour is enough. The incubate time response to the situation of
cell’s type and concentration. You can try to read the result after 0.5 hour, 1hour, 2 hours and 4
hours solely at first time, then chose a proper time for next step.
5. Read absorbance at 450nm. If there’s no 450nm filter, use 420-480nm instead. During dual
wavelength spectrophotometry, you may choose wavelength longer than 600nm.
Note
1. If cell culture time is too long, please pay attention to the evaporation issue. Avoid using the outmost
wells, add PBS buffer, water or culture fluid instead; or place 96 wells near by the water in incubator.
2. This kit bases on the catalytic reaction of dehydrogenates. If there are too many reducing agents in
the system ready to detect, such as some antioxidant which may interrupt the result, you should
remove them first.
3. Make sure that there’s no bubble in any well before use the ELISA reader, or it will interrupt the
result.
4. Please wear transparent gloves when operate.