Cell Counting Kit-8 Cell Proliferation Detection Kit

Cell Counting Kit-8 (CCK-8) Cell Proliferation Kit

Product Code: C6005M

Product specification: 500T

Storage conditions:4℃Store in a dark place for long-term storage-20℃，The expiration date can be found on the outer packaging.

Product Introduction

Cell Counting Kit-8abbreviationCCK8(orWST-8）A reagent kit is a type based onWST-8(Chemical name:2-(2-methoxy-4-Nitrophenyl group)-3-(4-Nitrophenyl group)-5-(2,4-Disulfobenzene)-2H-The widely used colorimetric reaction of tetrazolium monosodium salt is a rapid and highly sensitive detection kit for cell proliferation and cytotoxicity.

WST-8Working principle: In the presence of electron coupling reagents, it can be reduced by dehydrogenases in mitochondria to produce highly water-soluble orange yellow formamide（formazan）. The depth of color is directly proportional to cell proliferation and inversely proportional to cell toxicity. Using an enzyme-linked immunosorbent assay (ELISA) reader450 nmMeasurement at wavelengthODValue indirectly reflects the number of live cells.

WST-8Yes, it isMTTAn upgraded alternative product, andMTTOr otherMTTSimilar products such asXTTTheMTSCompared to others, there are obvious advantages. First,MTTThe formazan produced by the reduction of some dehydrogenases in mitochondria is not water-soluble and requires a specific solution to dissolve; AndWST-8TheXTTAndMTSThe generated formaldehyde is water-soluble, which eliminates the need for subsequent dissolution steps. Secondly,WST-8The generated nail ratioXTTAndMTSThe generated nail polish is more easily dissolved. againWST-8Compared toXTTAndMTSMore stable, making the experimental results more stable. Besides,WST-8WithMTTTheXTTCompared to linear range, it has a wider range and higher sensitivity.

CCKThe method is widely used in drug screening, cell proliferation assay, cytotoxicity assay, tumor drug sensitivity test, and activity detection of biological factors.

Usage:

1. Cell viability testing

（1）In96 Inoculate cell suspension into the orifice plate（100 μL/Hole), it is recommended to set it8 Cell concentration gradient, set for each concentration6 Repeat cell plating, for example, according to the number of cells0/312.5/625/1250/2500/5000/10000/20000 cells/96Lay the orifice plate. Place the culture plate in the incubator and incubate overnight（37℃,5% CO2）.

（2）Add to each hole10 μL CCKSolution (be careful not to generate bubbles in the pores, they will affect)ODThe reading of the value. Note: If there is precipitation in the product, it can be37℃Water bath treatment does not affect usage.

（3）Incubate the culture plate in the incubator0.5 - 4 hNote: The initial experiment can be conducted on0.5、1、2 And4 hAfterwards, use an enzyme-linked immunosorbent assay (ELISA) reader to detect each sample, and select a time point with a suitable absorbance range for subsequent experiments.

（4）Measure with an enzyme-linked immunosorbent assay (ELISA) reader450 nmAnd600 nmThe absorbance at the location where the background interference of the orifice plate is eliminated. （5）If not determined temporarilyODValue can be added to each hole10 μL 0.1 MTheHClSolution or1% w/v SDSSolution and store the culture plate at room temperature in the dark.24 hInternal measurement, absorbance will not change.

2. Cell proliferation toxicity testing

（1）In96 Inoculation in orifice plate100 μLThe cell suspension is recommended to be set up8 Cell concentration gradient, set for each concentration6 Repeat cell plating, for example, according to the number of cells0/312.5/625/1250/2500/5000/10000/20000 cells/96Lay the orifice plate. Place the culture plate in the incubator and incubate overnight（37℃, 5%CO2）

（2）Add to the culture plate1 - 10 μLSpecific drug stimulation.

（3）Incubate in the incubator for a period of time (e.g.:6、12、24 Or48 h）.

（4）Add to each hole10 μL CCKSolution (be careful not to generate bubbles in the pores, they will affect)ODThe reading of the value. Note: If there is precipitation in the product, it can be37℃Water bath treatment does not affect usage.

（5）Incubate the culture plate in the incubator0.5 – 4 hNote: The initial experiment can be conducted on0.5、1、2 And4 hAfterwards, use an enzyme-linked immunosorbent assay (ELISA) reader to detect each sample, and select a time point with a suitable absorbance range for subsequent experiments.

（6）Measure with an enzyme-linked immunosorbent assay (ELISA) reader450 nmAnd600 nmThe absorbance at the location where the background interference of the orifice plate is eliminated.

（7）If not determined temporarilyODValue can be added to each hole10 μL 0.1 MTheHClSolution or1% w/v SDSSolution, cover the culture plate and store it at room temperature in the dark.24 hInternal measurement, absorbance will not change.

Note: If the substance to be tested has oxidizing or reducing properties, it can be addedCCKPreviously, replace the fresh culture medium (remove the culture medium, wash the cells twice with the culture medium, and then add new culture medium) to remove the drug effect. Of course, in cases where the drug has a relatively small impact, the blank absorption after adding the drug to the culture medium can be directly deducted without changing the culture medium.

3. Calculation formula

cell viability=[(As-Ab)/(Ac-Ab)]xInhibition rate=[(Ac-As)/(Ac-Ab)]xAs:Experimental hole(Culture medium containing cellsCCK-8The drug to be tested)The absorbance ofAc:Comparison hole(Culture medium containing cellsCCK-8There are no drugs to be tested)The absorbance ofAb:Blank hole(Culture medium without cells and test drugsCCK-8)The absorbance of

Precautions

1. Before use, please centrifuge the product instantly to the bottom of the tube before conducting subsequent experiments.

2. use96During cell plating, if the cultivation time is long, attention must be paid to evaporation issues. Suggest abandoning the surrounding area and adding the same amount insteadPBSWater or culture medium.

3. Before using an enzyme-linked immunosorbent assay (ELISA) reader for detection, it is necessary to ensure that there are no bubbles in each well, otherwise it may interfere with the measurement.

4. This product is for scientific research only and should not be used for clinical diagnosis or treatment, food or medicine, or stored in ordinary residential areas.

5. For your safety and health, please wear lab coats and disposable gloves when operating.