A cell sample viability detection kit and detection method based on mass spectrometry flow cytometry technology

Abstract

This invention discloses a cell sample viability detection kit and method based on mass spectrometry flow cytometry. The cell sample viability detection kit based on mass spectrometry flow cytometry includes separately packaged solutions C, P, Pd, F, M, and D; solution C includes bovine serum albumin and phosphate buffer; solution P includes phosphate buffer; solution Pd includes palladium dichloride and double-distilled water. The effective component of palladium dichloride, palladium, has the following natural isotope abundances: isotope 102 The relative abundance of Pd was 1.02%. 104 Pd was 11.14%. 105 Pd is 22.33%. 106 Pd is 27.33%. 108 Pd was 26.46%. 110 Pd is 11.72%; Solution F includes paraformaldehyde and phosphate buffer; Solution M includes methanol; Solution D includes... 191 / 193 Ir, paraformaldehyde, and phosphate buffer. This invention's kit features high detection sensitivity, reduced detection interference, improved experimental efficiency, and reliable results.

Description

Technical Field

[0001] This invention belongs to the field of mass spectrometry flow cytometry detection technology, and relates to a cell sample activity detection kit and detection method based on mass spectrometry flow cytometry detection technology. Background Technology

[0002] Mass cytometry is a high-throughput single-cell analysis technique that combines flow cytometry with mass spectrometry. By using metal isotope-labeled antibodies or probes, mass cytometry can simultaneously detect dozens of different biomarkers at the single-cell level. Compared to traditional fluorescence flow cytometry, mass cytometry offers higher multiplexing capabilities and less signal overlap, demonstrating significant advantages in the in-depth analysis of complex samples. In flow cytometry, dead cells nonspecifically adsorb antibodies, leading to abnormal staining and causing significant detection errors, even masking key biological signals. Therefore, effectively identifying and eliminating interference from dead cells can significantly improve the accuracy and reproducibility of detection data, thus more accurately reflecting the physiological state of the cell population in the sample. Currently, in the field of cell viability detection, traditional methods mainly rely on dyes or fluorescently labeled reagents, such as propidium iodide (PI) and Annexin V / PI double staining. While these methods have been widely applied to fluorescence flow cytometry platforms, their limitations are becoming increasingly apparent, including the toxicity of the dyes themselves, severe signal spectral overlap, and interference with other labeled channels in multiplex detection. More importantly, these dyes cannot be used in mass flow cytometry platforms because they do not contain metal tags suitable for mass spectrometry detection, making it difficult to meet the platform's technical requirements for reagent metal labeling and signal resolution.

[0003] To meet the detection requirements of mass spectrometry platforms, cisplatin has been introduced to distinguish between live and dead cells. This method utilizes cisplatin's ability to penetrate the cell membrane of dead cells and form covalent bonds with intracellular proteins, thereby labeling the cell's viability. However, the use of cisplatin still faces a series of challenges, including complex preparation processes, poor reagent stability, and some toxicity to cells. Furthermore, with the rapid development of mass cytometry platforms, existing viability detection methods can no longer meet the demands of large-scale sample processing, standardized data generation, and high-throughput research. There is an urgent need to develop a cell viability detection solution with the following characteristics: 1) optimized specifically for mass spectrometry platforms, possessing high adaptability; 2) a simple labeling process suitable for automated workflows; 3) high reagent stability and low toxicity, ensuring cell integrity; 4) clear and highly sensitive detection signals, supporting multi-channel parallel readings; and 5) good commercialization potential, facilitating widespread deployment in clinical and research settings.

[0004] Therefore, developing a novel, stable, easy-to-operate, and highly specific cell viability detection kit and method has become an important breakthrough for promoting the development and application of mass cytometry technology. Summary of the Invention

[0005] To overcome the difficulty in accurately distinguishing between live and dead cells and effectively eliminating interference from dead cells in existing technologies, while improving the reproducibility and scalability of detection techniques and meeting the adaptation needs of multiple sample types and research objectives, this invention provides a cell viability detection kit and method based on mass spectrometry flow cytometry. This approach features excellent platform compatibility, ease of operation, and detection stability, significantly improving the accuracy and data quality of live cell identification in mass spectrometry flow cytometry. It provides strong technical support for fields such as immunology, oncology, and stem cell research, and also possesses broad prospects for commercialization and clinical translation.

[0006] This invention proposes a method for distinguishing between live and dead cells using palladium isotope mixture labeling technology and mass spectrometry analysis, thereby improving the sensitivity and accuracy of detection. This invention aims to further optimize and validate cell viability detection methods based on palladium isotope labeling technology, improve detection sensitivity and accuracy by refining reagent kit preparation and experimental procedures, and expand its applications in cell biology research, drug screening, and toxicology.

[0007] The present invention provides a cell sample activity detection kit based on mass spectrometry flow cytometry detection technology, comprising individually packaged solutions C, P, Pd, F, M, and D;

[0008] Solution C includes bovine serum albumin (full components, BSA) and phosphate buffered saline (PBS).

[0009] The P solution includes a phosphate buffer solution;

[0010] The Pd solution comprises palladium dichloride (PdCl2) and double-distilled water (ddH2O). The effective component of palladium dichloride, palladium, has the following natural isotopic abundances: Isotope 102 The relative abundance of Pd was 1.02%. 104 Pd was 11.14%. 105 Pd is 22.33%. 106 Pd is 27.33%. 108 Pd was 26.46%. 110 Pd was 11.72%;

[0011] The F solution includes paraformaldehyde (PFA) and phosphate buffer solution;

[0012] The M liquid includes methanol;

[0013] The D liquid includes 191 / 193 Ir, paraformaldehyde, phosphate buffer.

[0014] In the cell sample viability assay kit described above, solution C is a 1X phosphate buffer containing 2.5~5 mg / mL (specifically 5 mg / mL) bovine serum albumin.

[0015] In the cell sample viability assay kit described above, the P solution is 1X phosphate buffer.

[0016] In the cell sample viability assay kit described above, the Pd solution is a 500 μM double-distilled palladium dichloride aqueous solution.

[0017] In the cell sample viability assay kit described above, solution F is a phosphate buffer containing 1.6% paraformaldehyde by volume.

[0018] In the aforementioned cell sample viability assay kit, solution D has a final concentration of 125 nM. 191 / 193 Ir, its solvent is a phosphate buffer containing 1.6% paraformaldehyde by volume.

[0019] In this invention, the cell sample viability detection kit based on mass spectrometry flow cytometry detection technology includes individually packaged solutions C (100-200 mL), P (100-200 mL), Pd (2-5 mL), F (100-200 mL), M (50-100 mL), and D (2-5 mL). The volume range of each solution is sufficient for 50 cell viability detection kit tests.

[0020] The present invention also provides a method for detecting cell sample activity using the above-mentioned cell sample activity detection kit, comprising the following steps:

[0021] 1) Cell preparation: Wash the cell samples with the C solution and the P solution respectively, and then centrifuge to remove the supernatant;

[0022] 2) Palladium activity staining: Dilute the Pd solution with the P solution to obtain a diluted working solution. Resuspend the cells from step 1) in the diluted working solution and incubate at room temperature to obtain palladium activity stained cells.

[0023] 3) Add the palladium-stained cells to the C solution and centrifuge to remove the supernatant. Wash the cells with the C solution and P solution respectively, and then centrifuge to remove the supernatant.

[0024] 4) Fixation: The cells treated in step 3) were added dropwise to the F solution while vortexing, and incubated at room temperature;

[0025] 5) After the cells are fixed in step 4), add the C solution and centrifuge to remove the supernatant. Wash the cells with the C solution and the P solution respectively, and then centrifuge to remove the supernatant.

[0026] 6) Membrane rupture: After treatment in step 5), the cells are added dropwise to the M solution while vortexing, and incubated.

[0027] 7) After the cells in step 6) are perforated, add solution C and centrifuge to remove the supernatant. Wash with solution C and solution P in sequence, and then centrifuge to remove the supernatant.

[0028] 8) Cell DNA staining: Using the D solution, the cells treated in step 7) are pipetted and incubated at room temperature;

[0029] 9) Rinsing and instrumentation: Rinse the cells treated in step 8) with the P solution, rinse with double-distilled water, transfer to the flow cytometer tube of the mass spectrometer, count, and then detect the palladium isotope signal intensity of each cell.

[0030] 10) Process the palladium isotope signal intensity detection data obtained in step 9 to obtain the activity of the cell sample.

[0031] In the above method, in step 2), the P solution dilutes the Pd solution to 100 nM to 2.5 μM, preferably to 500 nM;

[0032] The number of cell samples is 2 to 3 million, and the amount of Pd solution added to the cells in step 1) can be 0.5 to 1 ml, specifically 1 ml;

[0033] In step 3), the amount of C solution and P solution added to the palladium-stained cells obtained in step 2) can be 2~4 mL, specifically 2 mL;

[0034] In step 4), the amount of solution F added to the cells obtained after step 3) can be 1~2mL, specifically 1mL;

[0035] In step 5), the amount of C solution and P solution added to the cells fixed in step 4) can be 2~4mL, specifically 2mL;

[0036] In step 6), the amount of M solution added to the cells treated in step 5) can be 1~2mL, specifically 1mL;

[0037] In step 7), the amount of C solution and P solution added to the cells after cell membrane rupture in step 6) can be 2~4mL, specifically 2mL;

[0038] In step 8), the amount of D solution added to the cells after the treatment in step 7) can be 0.5~1ml, specifically 1mL.

[0039] In the above method, the centrifugation rate in steps 1), 3), 5), and 7) can be 300~500×g, specifically 400×g, and the centrifugation time can be 5~15 minutes, specifically 10 minutes.

[0040] In the above method, in step 2), the incubation time can be 3 to 10 minutes, specifically 5 minutes;

[0041] In step 4), the incubation time can be 10-20 minutes, specifically 10 minutes; the incubation temperature is room temperature, specifically 10-30℃.

[0042] In step 6), the incubation temperature can be 4°C, and the time can be 20-30 minutes, specifically 20 minutes;

[0043] In step 8), the incubation time can be 60 to 90 minutes, specifically 60 minutes;

[0044] In step 9), the number of times the P solution is used for rinsing can be 2 to 3 times, specifically 2 times; the number of times the double-distilled water is used for rinsing can be 2 to 3 times, specifically 2 times.

[0045] In the above method, step 10) includes the palladium isotope signal intensity detection data. 102 Pd, 104 Pd, 105 Pd, 106 Pd, 108 Pd and 110 The signal intensity of at least one palladium isotope channel in Pd was analyzed using mass spectrometry flow cytometry to determine the cell activity status.

[0046] In this invention, the collected data is flow cytometry mass spectrometry information at the single-cell level, and the detected data includes the signal intensity of at least one palladium isotope channel. The palladium isotope signal intensity is positively correlated with cell membrane permeability; the higher the signal intensity, the stronger the cell membrane permeability, and thus the lower the cell's activity state. Therefore, for cell samples with significant differences in activity state, the signal intensity of a single isotope channel can be used to directly determine the activity state, which is efficient and practical, as shown in Example 1. However, when the differences in cell activity state are not obvious or the boundaries are blurred, the determination result of a single isotope channel may be uncertain. To improve the accuracy and robustness of the analysis, this invention further provides a multi-isotope signal synergistic analysis method: based on the collection of signals from two or more palladium isotope channels, their relative intensity, trend consistency, and distribution pattern are comprehensively analyzed to enhance the ability to identify cell activity state, as shown in Example 2. This method is particularly suitable for the activity determination of samples with high heterogeneity or critical states, improving analytical accuracy while taking into account flexibility and adaptability, which is one of the important technical features of this invention. In specific implementation, the palladium isotope channel can be selected from... 102 Pd, 104 Pd, 105 Pd, 106 Pd, 108 Pd and 110 Pd.

[0047] The present invention has the following beneficial effects:

[0048] 1. Improved detection sensitivity: This invention utilizes the unique physical properties of palladium isotopes to improve the sensitivity of cell viability detection through mass spectrometry analysis, enabling precise differentiation between live and dead cells.

[0049] 2. Reduced detection interference: The reagent kit of this invention uses palladium isotope labeling reagent (such as PdCl2), which is easy to prepare and high-purity reagents are more readily available, solving the problem of complex preparation of cisplatin reagent; palladium isotope labeling is non-toxic to cells, can better preserve the physiological state of cells, reduce the interference of traditional dyes or fluorescent labels on cells, and improve the accuracy of detection results.

[0050] 3. Optimized detection process: The method of this invention is simple and applicable to different types of cells. It can complete cell viability detection in a short time and improve experimental efficiency.

[0051] 4. Provides reliable data: Through precise analysis of mass spectrometry data, this invention provides reliable cell activity data, which can be applied to multiple fields such as cell biology research, drug screening, and toxicology.

[0052] In summary, this invention has significant technical advantages and broad application prospects in the field of cell viability detection. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of the reagent kit and analytical method for detecting cell sample viability based on mass spectrometry flow cytometry.

[0054] Figure 2 Cell viability was measured using different concentrations of palladium dichloride solution after adding 30% heat-inactivated Jurkat cells.

[0055] Figure 3 The natural abundance of different palladium isotopes and the signal intensity of palladium isotopes in dead cells at different concentrations (L: low, 100 nM PdCl2, M: medium, 500 nM PdCl2, H: high, 2.5 μM PdCl2) and the correlation between natural abundance and signal intensity were shown.

[0056] Figure 4 To use a single isotope in peripheral blood mononuclear cell samples ( 108 Pd) and the combined use of multiple isotopes ( 106 Pd / 108 Pd / 110 Pd) analysis was used to analyze the differences in cell sample activity. Detailed Implementation

[0057] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following specific embodiments.

[0058] The embodiments provided below are not intended to limit the scope of this invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to this invention by those skilled in the art in conjunction with existing common knowledge also fall within the scope of protection claimed by this invention.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0060] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0061] The present invention provides a cell sample activity detection kit based on mass spectrometry flow cytometry detection technology, comprising individually packaged solutions C, P, Pd, F, M, and D;

[0062] Solution C includes bovine serum albumin (full components, BSA) and phosphate-buffered saline (PBS).

[0063] Solution P includes phosphate buffer;

[0064] Pd solution includes palladium dichloride (PdCl2) and double-distilled water (ddH2O);

[0065] Solution F includes paraformaldehyde (PFA) and phosphate buffer;

[0066] M liquid includes methanol;

[0067] D liquid includes 191 / 193 Ir, paraformaldehyde, phosphate buffer.

[0068] In the following examples, the cell sample viability assay kit based on mass spectrometry flow cytometry detection technology includes the following individually packaged solutions:

[0069] Solution C: 1X phosphate buffer containing 5 mg / mL bovine serum albumin;

[0070] P solution: 1X phosphate buffer;

[0071] Pd solution: 500 μM palladium dichloride (commercially available from Sigma-Aldrich, catalog number 323373) double-distilled aqueous solution;

[0072] Solution F: Phosphate buffer containing 1.6% paraformaldehyde by volume;

[0073] Solution D: Final concentration 125 nM 191 / 193 Ir (commercially purchased from Standard Biotools, catalog number 201192A), its solvent is phosphate buffer containing 1.6% paraformaldehyde by volume.

[0074] This invention relates to a cell sample viability assay kit based on mass spectrometry flow cytometry detection technology, comprising individually packaged solutions C (100-200 mL), P (100-200 mL), Pd (2-5 mL), F (100-200 mL), M (50-100 mL), and D (2-5 mL). The volume range of each solution is sufficient for 40-60 cell viability assays.

[0075] Example 1

[0076] A method for detecting cell sample activity using a mass cytometry-based cell sample activity assay kit is described below:

[0077] Step 1: Cell Preparation

[0078] 1. Jurkat Clone E6-1 cells (commercially purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd., product catalog number BFN60700175) were cultured to a number of approximately 3 million, and cell counting was performed;

[0079] 2. Select 30% of the cells and inactivate them in a 55℃ water bath for 1 hour;

[0080] 3. Mix the inactivated cells with the remaining cells thoroughly;

[0081] 4. Centrifuge to remove the supernatant;

[0082] 5. Add 2 mL of solution C to the centrifuge tube, centrifuge at 400×g for 10 minutes, and discard the supernatant;

[0083] 6. Wash the cells again with 2 mL of P solution, centrifuge at 400×g for 10 minutes, and discard the supernatant;

[0084] Step 2, Palladium active staining:

[0085] 1. The washed cells were resuspended in 1 mL of palladium solutions of different concentrations (100 nM, 500 nM, 2.5 μM);

[0086] 2. Incubate at room temperature (25℃) for 5 minutes to ensure that the palladium isotope fully penetrates the cells and completes the labeling;

[0087] 3. Add 2 mL of solution C to dilute the staining solution and terminate the staining reaction. Centrifuge at 400×g for 10 minutes and discard the supernatant.

[0088] 4. Resuspend the cells in 2 mL of C solution, centrifuge at 400×g for 10 minutes, and discard the supernatant;

[0089] 5. Resuspend the cells in 2 mL of P solution and centrifuge again at 400×g for 10 minutes to remove unbound palladium isotopes;

[0090] Step 3: Fix:

[0091] 1. Add 1 mL of F solution dropwise to the cell suspension while vortexing to ensure uniform fixation;

[0092] 2. Incubate at room temperature for 10 minutes;

[0093] Step 4: Post-fixation processing:

[0094] 1. Add 2 mL of solution C, centrifuge at 400×g for 10 minutes, and remove the supernatant;

[0095] 2. Resuspend the cells in 2 mL of C solution, centrifuge at 400×g for 10 minutes, and remove the supernatant;

[0096] 3. Resuspend the cells in 2 mL of P solution, centrifuge again at 400×g for 10 minutes, and remove the supernatant;

[0097] Step 5: Break the membrane:

[0098] 1. Add 1 mL of M solution dropwise to the cell suspension while vortexing to ensure complete membrane rupture;

[0099] 2. Incubate at 4℃ for 20 minutes;

[0100] Step Six: Post-Membrane Breakage Processing

[0101] 1. Add 2 mL of solution C, centrifuge at 400×g for 10 minutes, and remove the supernatant;

[0102] 2. Resuspend the cells in 2 mL of C solution, centrifuge at 400×g for 10 minutes, and remove the supernatant;

[0103] 3. Resuspend the cells in 2 mL of P solution, centrifuge again at 400×g for 10 minutes, and remove the supernatant;

[0104] Step 7: Cell DNA Staining

[0105] 1. Resuspend the treated cells in 1 ml of solution D and incubate at room temperature for 60 minutes to ensure that the DNA inside the cells is fully stained;

[0106] 2. After staining, wash the cells twice with 2 mL of P solution and twice with 2 mL of double-distilled water to remove excess staining solution and prepare for subsequent mass spectrometry analysis.

[0107] Step 8: Mass spectrometry analysis:

[0108] 1. Suspend the treated cells in an appropriate amount of double-distilled water, preferably (0.5~1)×10⁻⁶. 6 Concentration per cell / mL;

[0109] 2. Cell analysis using a mass spectrometer: Recommended operating parameters for the mass spectrometer (using Helios MassCytometer, Standard BioTools as an example) are: flow rate set to 30 μL / min, event rate between 250-350 events / s, mass spectrometry resolution based on 159 Tb, and sensitivity exceeding 600,000 counts / 159 Tb; argon flow rate set to 20 L / min; background subtraction and data normalization performed during data acquisition; and EQ applied. TM Four-element calibration beads (1 / 10V / V); detector voltage set to -2880 V, operating temperature maintained at 22 °C ± 2 °C, sample introduced using a pneumatic round-bottom tube to obtain optimal signal;

[0110] 3. During the analysis, the mass spectrometer detects the mass-to-charge ratio (m / z) of the palladium isotope and records its signal intensity, thereby determining the palladium isotope signal intensity of each cell;

[0111] Step Nine, Data Processing:

[0112] 1. Analyze the obtained mass spectrometry data using mass spectrometry data processing software (e.g., FlowJo or Cytobank);

[0113] 2. Based on the intensity and distribution of palladium isotope signals, cells are divided into two categories: living cells and dead cells.

[0114] 3. By combining the signal intensities of different palladium isotopes, the viability of the cells can be further confirmed.

[0115] In this invention, the Jurkat cells prepared in step 1) of Example 1 above had a viability of 70%, and were compared using trypan blue staining and the sample viability detection kit of this invention. The results showed that the sample viability detection using low, medium, and high concentrations (100 nM, 500 nM, 2.5 μM) of PdCl2 of this invention was comparable to that using traditional trypan blue staining. Among these, the medium concentration (500 nM) of PdCl2 showed better separation of live and dead cells and a higher signal-to-noise ratio. Furthermore, the experimental results showed differences in staining effects with different palladium isotopes (…). Figure 2 Data analysis showed that the signal intensity of different palladium isotopes in dead cells was significantly correlated with their natural abundance. Figure 3 ).

[0116] Example 2

[0117] To enhance the application of this invention and further meet the needs of detecting complex cell samples beyond single cell lines, the following application extensions have been made using peripheral blood mononuclear cells as samples:

[0118] Step 1: Cell Preparation

[0119] 1. Count peripheral blood mononuclear cells (purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd., product catalog number P122042202C) to 2-3 million cells.

[0120] 2. Centrifuge to remove the supernatant.

[0121] 3. Add 2 mL of solution C to the centrifuge tube, centrifuge at 400×g for 10 minutes, and discard the supernatant.

[0122] 4. Wash the cells again with 2 mL of P solution, centrifuge at 400×g for 10 minutes, and discard the supernatant.

[0123] Step 2, Palladium active staining:

[0124] 1. Resuspend the washed cells in 1 mL of 500 nM palladium solution.

[0125] 2. Incubate at room temperature (25℃) for 5 minutes to ensure that the palladium isotope fully penetrates the cells and completes the labeling.

[0126] 3. Add 2 mL of solution C to dilute the staining solution and terminate the staining reaction. Centrifuge at 400×g for 10 minutes and discard the supernatant.

[0127] 4. Resuspend the cells in 2 mL of C solution, centrifuge at 400×g for 10 minutes, and discard the supernatant.

[0128] 5. Resuspend the cells in 2 mL of P solution and centrifuge again at 400×g for 10 minutes to remove unbound palladium isotopes.

[0129] Step 3: Fix:

[0130] 1. Add 1 mL of F solution dropwise to the cell suspension while vortexing to ensure uniform fixation.

[0131] 2. Incubate at room temperature for 10 minutes.

[0132] Step 4: Post-fixation processing:

[0133] 1. Add 2 mL of solution C, centrifuge at 400xg for 10 minutes, and remove the supernatant.

[0134] 2. Resuspend the cells in 2 mL of C solution, centrifuge at 400×g for 10 minutes, and remove the supernatant.

[0135] 3. Resuspend the cells in 2 mL of P solution, centrifuge again at 400×g for 10 minutes, and remove the supernatant.

[0136] Step 5: Break the membrane:

[0137] 1. Add 1 mL of M solution dropwise to the cell suspension while vortexing to ensure complete membrane rupture.

[0138] 2. Incubate at 4℃ for 30 minutes.

[0139] Step Six: Post-Membrane Breakage Processing

[0140] 1. Add 2 mL of solution C, centrifuge at 400×g for 10 minutes, and remove the supernatant.

[0141] 2. Resuspend the cells in 2 mL of C solution, centrifuge at 400×g for 10 minutes, and remove the supernatant.

[0142] 3. Resuspend the cells in 2 mL of P solution, centrifuge again at 400×g for 10 minutes, and remove the supernatant.

[0143] Step 7: Cell DNA Staining

[0144] 1. Resuspend the treated cells in 1 ml of solution D and incubate at room temperature for 60 minutes to ensure that the DNA inside the cells is fully stained;

[0145] 2. After staining, wash the cells twice with 2 mL of P solution and twice with 2 mL of double-distilled water to remove excess staining solution and prepare for subsequent mass spectrometry analysis; Step 8, Mass Spectrometry Analysis:

[0146] 1. Suspend the treated cells in an appropriate amount of double-distilled water, preferably (0.5~1)×10⁻⁶. 6 Concentration of cells / mL.

[0147] 2. Recommended operating parameters for the mass spectrometer (using Helios Mass Cytometer, Standard BioTools as an example): flow rate set to 30 μL / min, event rate between 250-350 events / s, mass spectrometry resolution based on 159Tb, sensitivity exceeding 600,000 counts / 159Tb; argon flow rate set to 20 L / min; background subtraction and data normalization performed during data acquisition; EQTM four-element calibration beads (1 / 10 v / v); detector voltage set to -2880V; operating temperature maintained at 22 °C ± 2 °C; sample introduced using a pneumatic round-bottom tube to obtain optimal signal.

[0148] 3. During the analysis, the mass spectrometer detects the mass-to-charge ratio (m / z) of palladium isotopes and records their signal intensity to determine the palladium isotope content of each cell.

[0149] Step Nine, Data Processing:

[0150] 1. Analyze the obtained mass spectrometry data using mass spectrometry data processing software (e.g., FlowJo or Cytobank).

[0151] 2. The palladium isotope signal of each cell was normalized, and the cells were divided into two categories, live cells and dead cells, according to the intensity and distribution of the palladium isotope signal.

[0152] 3. By combining the signal intensities of different palladium isotopes, the viability of the cells can be further confirmed.

[0153] In this invention, peripheral blood mononuclear cells prepared in step one of Example 2 above were subjected to comparative staining using the sample viability detection kit of this invention, and cell viability was analyzed using multiple palladium isotopes. The results showed that in complex cell samples, using a single isotope (…) Figure 4 (1) 108When analyzing Pd, the boundary between dead and living cells is not clear, so isotopes with higher natural abundance are used. Figure 4 (2) and (3) 108 Pd, 106 Pd and 110 Combined analysis of Pd can more accurately distinguish between dead and live cells, resulting in better cell viability detection. Figure 4 The results above show that the present invention can improve the accuracy and effectiveness of cell viability detection.

[0154] Through the above steps, the method of the present invention can accurately distinguish between live and dead cells using palladium isotope labeling and metal intercalation staining techniques without affecting cell viability, thereby improving the sensitivity and accuracy of cell viability detection. The specific operating methods and optimized parameters for each step are described in detail with reference to the accompanying drawings.

Claims

1. A method for detecting cell sample activity using a cell sample activity assay kit based on mass spectrometry flow cytometry, wherein the cell sample activity assay kit based on mass spectrometry flow cytometry includes separately packaged solutions C, P, Pd, F, M, and D; solution C includes bovine serum albumin and phosphate buffer; solution P includes phosphate buffer; solution Pd is a 500 μM double-distilled aqueous solution of palladium dichloride, wherein the abundance of the natural isotopes of palladium, the active ingredient in palladium dichloride, is as follows: isotope 102 The relative abundance of Pd was 1.02%. 104 Pd was 11.14%. 105 Pd is 22.33%. 106 Pd is 27.33%. 108 Pd was 26.46%. 110 The Pd value is 11.72%; the F solution comprises paraformaldehyde and phosphate buffer; the M solution comprises methanol; the D solution comprises... 191 / 193 Ir, paraformaldehyde, and phosphate buffer; including the following steps: 1) Cell preparation: Wash the cell samples with the C solution and the P solution respectively, and then centrifuge to remove the supernatant; 2) Palladium activity staining: Dilute the Pd solution with the P solution to obtain a diluted working solution. Resuspend the cells from step 1) in the diluted working solution and incubate at room temperature to obtain palladium activity stained cells. In step 2), the Pd solution is diluted to 100 nM to 2.5 μM using the P solution; 3) Add the palladium-stained cells to the C solution and centrifuge to remove the supernatant. Wash the cells with the C solution and P solution respectively, and then centrifuge to remove the supernatant. 4) Fixation: The cells treated in step 3) are added dropwise to solution F while vortexing, and incubated. 5) After the cells are fixed in step 4), add the C solution and centrifuge to remove the supernatant. Wash the cells with the C solution and the P solution in sequence, and then centrifuge to remove the supernatant. 6) Membrane rupture: After treatment in step 5), the cells are added dropwise to the M solution while vortexing, and incubated. 7) After the cells in step 6) are perforated, add the C solution and centrifuge to remove the supernatant. Wash with C solution and P solution in sequence, and then centrifuge to remove the supernatant. 8) Cell DNA staining: Using the D solution, the cells treated in step 7) are pipetted and incubated at room temperature; 9) Rinsing and instrumentation: Rinse the cells treated in step 8) with the P solution, rinse with double-distilled water, transfer to the flow cytometer tube of the mass spectrometer, count, and then detect the palladium isotope signal intensity of each cell. 10) Process the palladium isotope signal intensity detection data obtained in step 9) to obtain cell sample activity data; In step 10), the palladium isotope signal intensity detection data includes 102 Pd, 104 Pd, 105 Pd, 106 Pd, 108 Pd and 110 Signal intensity of at least one palladium isotope channel in Pd.

2. The method according to claim 1, characterized in that, Solution C is a 1X phosphate buffer containing 2.5~5 mg / mL bovine serum albumin.

3. The method according to claim 1 or 2, characterized in that, The P solution is a 1X phosphate buffer solution.

4. The method according to claim 1 or 2, characterized in that, The F solution is a phosphate buffer containing 1.6% paraformaldehyde by volume.

5. The method according to claim 1 or 2, characterized in that, The D solution has a final concentration of 125 nM. 191 / 193 Ir, its solvent is a phosphate buffer containing 1.6% paraformaldehyde by volume.

6. The method according to claim 1, characterized in that, The number of cell samples is 2 to 3 million, and the volume of diluted working solution added to the cells in step 1) is 0.5 to 1 ml; In step 3), the amount of C solution and P solution added to the palladium-stained cells obtained in step 2) is 2-4 mL; In step 4), the amount of solution F added to the cells obtained after treatment in step 3) is 1~2 mL; In step 5), the amount of C solution and P solution added to the cells fixed in step 4) is 2-4 mL; In step 6), the amount of the cells treated in step 5) added to the M solution is 1~2 mL; In step 7), the amount of C solution and P solution added to the cells after cell membrane rupture in step 6) is 2-4 mL; In step 8), the amount of D solution added to the cells after the treatment in step 7) is 0.5~1ml.

7. The method according to claim 1 or 6, characterized in that, In steps 1), 3), 5), and 7), the centrifugation rate is 300-500 × g, and the centrifugation time is 5-15 minutes. In step 2), the incubation time is 3-10 minutes; In step 4), the incubation time is 10-20 minutes; In step 6), the incubation temperature is 4°C and the time is 20-30 minutes; In step 8), the incubation time is 60-90 minutes; In step 9), the number of times the P solution is used for rinsing is 2 to 3 times, and the number of times the double-distilled water is used for rinsing is 2 to 3 times.

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