I would be very careful with accusations of academic fraud. I would suggest to first ask the authors for an explanation regarding the data interpretation. The best platform to do that is https://pubpeer.com/search?q=+33878182.

Thank you very much for your suggestions. I apologize for the inconvenience, but it seems that my Pubpeer account registration is consistently unsuccessful, possibly due to my Chinese IP address, even though I am using my university email. Prior to this, I had raised objections to the author of the paper because the data is just too abnormal. The flow cytometry graphical data is unremarkable with other cell proliferation methods, but when combined with CFSE staining, it's just too bizarre. However, the author adamantly argues back, even claiming that they followed the kit's instructions to the letter, with the procedure being cell culture stimulation for proliferation followed by CFSE staining for flow cytometry analysis. I will translate the descriptive part of this image for friends who will answer this question later for reference. Thank you again for your generous advice.

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Here is the original source of the data, which is a master's thesis by a graduate student of the corresponding author of the paper. Since it's a thesis written in Chinese, I've included screenshots of the problematic data along with the relevant reagents and procedural steps involved.

**Evidence Material Information and Detailed Explanation**

In Document, which serves as the data source, it is clearly stated where the CFSE detection reagent kit was obtained, as well as the staining and detection steps and the final experimental results. The following content is a screenshot and translation comparison of the reagent information and operational steps related to the paper.

1. On page 26 of the document (highlighted in yellow), it clearly shows the source information of CFSE dye:

**Yellow Text Content:**

Reagent Name, CFSE Cell Proliferation and Toxicity Detection Kit; Source, Beijing BioLab Technology Co., Ltd.

2. On page 27 of the document (highlighted in yellow), it clearly states the implemented operational steps:

**Yellow Text Content:**

1.4.3 Main Experimental Procedures

1.4.3.1 Dilute the CFSE cell labeling solution 10-fold with ultrapure water.

1.4.3.2 According to the number of cell samples to be detected, dilute the CFSE cell labeling solution 100-500 times, then dilute 2-8 times with PBS to prepare the staining working solution, with a final concentration equivalent to diluting the CFSE mother solution 200 times.

1.4.3.3 Resuspend the cells to be tested at a concentration of 10^7 cells/ml with the staining working solution in a centrifuge tube.

1.4.3.4 Incubate in a 37°C incubator away from light for 15-30 minutes.

1.4.3.5 After centrifugation, discard the supernatant, collect the cells, and wash the cells 1-2 times with L500 serum-free lymphocyte culture medium, then resuspend the cells in culture medium.

1.4.3.6 Take 500μl of the cell suspension and detect cell proliferation with a flow cytometer, the labeled cells exhibit green fluorescence.

3. On pages 47-48 of the document are the experimental results of CFSE detection of lymphocyte proliferation:

**Yellow Text Content:**

2.5 The Effect of IL-35 on the Proliferation of CD4+ T Lymphocytes

Using a gradient concentration (final concentration of 0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 500 ng/ml) of rhIL-35 to stimulate the culture of ex vivo expanded CD4+ T lymphocytes for 5 days, the CFSE staining detection of CD4+ T lymphocyte proliferation showed that rhIL-35 significantly inhibited the proliferation of CD4+ T lymphocytes at 10 ng/ml (25.92±3.75 vs 19.05±0.56), P < 0.05; 100 ng/ml (25.92±3.75 vs 14±2.76), P < 0.05; 500 ng/ml (25.92±3.75 vs 4.94±1.12), P < 0.001. There was no statistical difference in the 1 ng/ml treatment group (25.92±3.75 vs 23.4±2.8), P=0.40, see Figure 5.

The above is the reagent box information and operational and final data material used in Document.

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The information about the reagent box and the experimental operation steps displayed on the official website:

Company's official website address: https://www.bjbalb.com/index.htm

Search results for "CFSE Cell Proliferation and Toxicity Detection Kit" on the company's official website: https://www.bjbalb.com/product.aspx?kwd=HR8305

The information about the reagent box and the recommended experimental steps provided on the company's official website:

Cell Measurement

1. Dilute the CFSE cell labeling solution 10 times with pure water.

2. According to the number of cell samples to be detected, dilute the CFSE mother solution 100-500 times with the cell labeling solution, then dilute 2-8 times with PBS to prepare the staining working solution, with a final concentration equivalent to diluting the CFSE mother solution 200-4000 times. Different cells need to determine the optimal staining concentration according to the results of a preliminary experiment. If the fluorescence intensity is weak, the final concentration of CFSE can be appropriately increased. If the background is too high, the final concentration of CFSE can be appropriately reduced. Please explore the conditions to find the best concentration.

3. Resuspend the cells to be tested with the staining working solution to a cell concentration of approximately 10^7/ml.

4. Incubate in a 37°C incubator for 15-30 minutes.

5. After centrifugation, discard the supernatant, collect the cells, and wash the cells 1-2 times with PBS or serum-free medium, then resuspend the cells in PBS or serum-free medium.

6. Take 500μl of the cell suspension and detect with a flow cytometer or fluorescence microscope.

7. Subsequently, you can also culture according to the normal culture method of the cells. The marking effect can be directly observed under a fluorescence microscope, and the cell proliferation can be detected with a flow cytometer after a suitable period of culture, or used for other specific experimental purposes of cell fluorescence tracing. The labeled cells can also be transplanted into living animals and traced with fluorescence. The labeled cells exhibit green fluorescence.

It can be seen that the operation process and detection results in Document A show that the other party does not understand that CFSE is used to detect cell proliferation through fluorescence decay. The operation and data in Document A clearly indicate that they first stimulated T cell proliferation and then stained with CFSE, and then performed flow cytometry analysis after such operation. This is obviously wrong. @Sabine Strehl

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Sabine Strehl Thank you very much for your help. I have submitted my opinion in pubpeer, which may need to be reviewed before it can be displayed. After listening to your suggestions yesterday, I also found some streaming experts to analyze the problem, and it can be determined that the data is wrong. Thank you very much for your advice, thank you again.