Little talk:
I use CCK-8 (Cat.No.: HY-K0301) and the measured values are different each time. What is the reason? What substances will affect the determination of CCK-8? Is the sensitivity the same for different cells?
Recently, when I used the CCK-8 kit (MCE catalog number HY-K0301) for cell viability detection, I encountered the problem of large fluctuations in the test values.
I would like to ask you:
1. Differences in repeatability of test values
Under the same experimental conditions (such as cell density, culture time, and reagent addition), when the same cell sample is measured multiple times, the OD value fluctuation range can reach 10%-15%. What causes this difference?
2. Types of interfering substances
The literature mentions that some substances may react with CCK-8. I would like to know which common experimental reagents (such as antibiotics and transfection reagents) may significantly interfere with the test results? Is it necessary to wash the cells or replace the culture medium before the test?
3. Cell type-specific sensitivity
Are there any CCK-8 optimization schemes for different cell types (such as incubation time, reagent concentration)? For cells with low metabolic activity, is it necessary to extend the color development time or increase the cell inoculation amount?
[Troubleshooting Questions are selected from MCE customer consultation emails.]
Analysis of factors affecting CCK-8 (MCE, Cat.No.: HY-K0301) detection and result differences:
1. Interfering substances
Substances with reducing properties may affect the CCK-8 reaction, such as glucose solution containing vitamin C (usually low in culture medium). In addition, although phenol red can increase the blank absorbance, the effect can be eliminated by deducting the blank value, and serum components generally do not interfere with the determination.
2. Reasons for fluctuations in detection values
- Edge effect:
When cultured in an incubator, the outermost wells of the culture plate are prone to volume errors due to liquid evaporation. It is recommended that only culture medium be added to the outer wells as a blank control and not used for sample determination.
- Reagent mixing problem:
CCK-8 solution adheres to the well wall, which may cause uneven local concentration. It is recommended to tap the culture plate after adding the sample to promote mixing.
- Cell seeding density:
The deviation of the number of cells per well from the optimal range will cause errors. It is necessary to explore and optimize the seeding density in the density range of 1,000-100,000 cells/well in advance.
3. Cell type-specific sensitivity
- CCK-8 has different sensitivities to different cells.
- Suspension cells are usually more difficult to stain than adherent cells because their cell membranes have lower permeability to WST-8.
- Adherent cells can obtain higher absorbance 1-4 hours after adding CCK-8, while suspension cells can improve the detection signal by extending the incubation time or increasing the cell inoculation amount.
The answer to this question comes from MCE Technical Support.
How to contact us?
- Tel: 609-228-6898
- Fax: 609-228-5909
- E-mail: [email protected]
- Technical Support: [email protected]
- Address: 1 Deer Park Dr, Suite F, Monmouth Junction, NJ 08852, USA
- Badges
- Science topic
- Similar topics
- Biological Science
- Physiology
- Physiological Processes
- Stress