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The Data Doesn't Match? Correlation Analysis of Cell Cycle & CCK-8 Results

Source: Elabscience®Published: Jan 16,2024

Have you ever had such an experience? Have done experiments for more than half a year, CCK-8, cell cycle, cell apoptosis, ELISA...... Experiments have been done a lot, when analyzing the results, found the data are all not match.

  • CCK-8 detection after treatment showed that there was a significant inhibitory effect on proliferation
  • However, the apoptosis detection showed that the apoptosis rate decreased after treatment
  • There was no change in cell cycle

Don't worry, the truth is often hidden in these seemingly “conflicting” experimental results, maybe there is nothing wrong with your results.

To analyze the relationship between these two experimental results, we first need to know exactly what does each experimental result means?

Some people may think the result is too simple, CCK-8 indicates the rate of cell proliferation, and cell cycle detection results indicate the proportion of each stage in the cell cycle. To this, I can only say: right, but not entirely right.

To be precise, CCK-8 experimental result measures the total dehydrogenase activity in a sample, and cell cycle experimental result measures the nucleic acid content of each cell.

Fig. 1 Schematic diagram of the principle of CCK-8 detection
Fig. 2 Schematic diagram of cycle detection principle

Only by looking at our experimental results from this level can we find the root of the problem.

According to the experimental principle, we can know that the change of OD value detected by CCK-8 may be caused by the following reasons:

Increased OD

Decreased OD

Increased cell number

Decreased cell number

Increased cell viability

Decreased cell viability

A reducing agent is present in the experimental system

Oxidizing agents are present in the experimental system

The reagent is yellow or cloudy when added

Metal ions are present in the experimental system

Similarly, the abnormality of cell cycle detection can also be explained by the experimental principle. In addition to the DNA in the nucleus, nucleic acid also contains a variety of substances such as RNA, which can bind to PI and thus affect the results of the analysis.

Before analyzing the relationship between CCK-8 and cell cycle, we first need to exclude the result changes caused by abnormal causes. For example:

  • The sample contains reductant oxidants/metal ions/colored-reagents, etc. Before CCK-8 detection, the liquid should be exchanged first;
  • During cell cycle detection, pay attention to control the cell inoculation density, and the cell density should not be too high when culturing to avoid contact inhibition between cells, which may result in a large number of cells staying in the G0 phase
  • RNase should be added during cell cycle detection to avoid the influence of RNA on the results.

After the above work is completed, let's take a look at the relationship between CCK-8 and cell cycle changes, and thepossible reasons of these changes:

CCK-8

Cell cycle

Reference Cause

Inhibition of proliferation

The G0/G1 phase increases

Drugs inhibit DNA replication

G2/M phase increases

Drugs inhibit mitosis (such as mitomycin,etc.)

S-phase increases

The drug caused G2/M phase arrest and continued treatment resulted in DNA fragmentation and partial nucleic acid leakage

Promotion of proliferation

G0/G1 phase increases

Excessive cell inoculation density

The G2/M phase increases

Promotes cell proliferation normally

S-phase increases

Promotes cell proliferation normally

Reagents relevant to this phase of the experiment

Cell Cycle Assay Kit (Red Fluorescence) E-CK-A351

Cell Cycle Assay Kit (Green Fluorescence) E-CK-A352

Cell Cycle Assay Kit (Blue Fluorescence) E-CK-A353

Enhanced cell viability assay kit (CCK-8) E-CK-A362

For more Flow Cytometry related questions, please visit the Flow Cytometric analysis, or consult the technical support on the right side of the page ~

Elabscience® Flow Cytometry Antibodies Features (←Click to view)

Multi-color optional: up to 13 fluorescent markers, suitable for more experimental protocols

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