Search

CN-120628954-B - Cell sample activity detection kit and detection method based on mass spectrometry detection technology

CN120628954BCN 120628954 BCN120628954 BCN 120628954BCN-120628954-B

Abstract

The invention discloses a cell sample activity detection kit and a detection method based on a mass spectrometry detection technology. The invention discloses a cell sample activity detection kit based on mass spectrometry detection technology, which comprises separately packaged C liquid, P liquid, pd liquid, F liquid, M liquid and D liquid, wherein the C liquid comprises bovine serum albumin and phosphate buffer solution, the P liquid comprises phosphate buffer solution, the Pd liquid comprises palladium dichloride and double distilled water, the palladium element serving as an effective component in the palladium dichloride has the natural isotope abundance of 1.02% of isotope 102 Pd, 11.14% of 104 Pd, 22.33% of 105 Pd, 27.33% of 106 Pd, 26.46% of 108 Pd and 11.72% of 110 Pd, the F liquid comprises paraformaldehyde and phosphate buffer solution, the M liquid comprises methanol, and the D liquid comprises 191/ 193 Ir, paraformaldehyde and phosphate buffer solution. The kit has the characteristics of high detection sensitivity, reduced detection interference, improved experimental efficiency and reliable results.

Inventors

  • HAN GUOJUN
  • HUANG PEIQING
  • ZHAO YANG

Assignees

  • 北京大学

Dates

Publication Date
20260505
Application Date
20250710

Claims (7)

  1. 1. A cell sample activity detection method adopting a cell sample activity detection kit based on a mass spectrometry detection technology comprises a C solution, a P solution, a Pd solution, an F solution, an M solution and a D solution which are packaged independently, wherein the C solution comprises bovine serum albumin and a phosphate buffer solution, the P solution comprises a phosphate buffer solution, the Pd solution is a double-distilled aqueous solution of palladium dichloride with the concentration of 500 mu M, the palladium element serving as an effective component in the palladium dichloride has the following natural isotope abundance that the relative abundance of isotope 102 Pd is 1.02%, 104 Pd is 11.14%, 105 Pd is 22.33%, 106 Pd is 27.33%, 108 Pd is 26.46%, 110 Pd is 11.72%, the F solution comprises paraformaldehyde and the phosphate buffer solution, the M solution comprises methanol, and the D solution comprises 191/193 Ir, paraformaldehyde and the phosphate buffer solution, and the following steps are included: 1) Cell preparation, namely washing a cell sample by using the solution C and the solution P respectively, and centrifuging to remove the supernatant; 2) The Pd liquid is diluted by the P liquid to obtain diluted working liquid, and the cells in the step 1) are resuspended in the diluted working liquid and incubated at room temperature to obtain palladium-activity-dyed cells; In the step 2), the Pd solution is diluted to 100 nM-2.5 mu M by using the P solution; 3) Adding the solution C into the palladium-activity-stained cells, centrifuging, removing supernatant, washing by using the solution C and the solution P respectively in sequence, and centrifuging to remove supernatant; 4) Fixing, namely dropwise adding the F solution into the cells treated in the step 3) while vortex oscillation, and incubating; 5) Adding the C solution into the cells fixed in the step 4), centrifuging, removing supernatant, washing by using the C solution and the P solution in sequence, and centrifuging to remove supernatant; 6) Membrane rupture, namely dropwise adding the M solution into the cells treated in the step 5) while vortex oscillation, and incubating; 7) Adding the C solution into the cells subjected to the membrane rupture in the step 6), centrifuging to remove supernatant, washing by using the C solution and the P solution in sequence respectively, and centrifuging to remove the supernatant; 8) Staining cell DNA, namely blowing the cells treated in the step 7) by using the solution D, and incubating at room temperature; 9) Rinsing, detecting on the machine, namely rinsing the cells treated in the step 8) by using the P liquid, transferring the cells to a flow tube of a mass spectrometry flow cytometer after rinsing by using double distilled water, and detecting the palladium isotope signal intensity of each cell on the machine after counting; 10 Processing the palladium isotope signal intensity detection data obtained in the step 9) to obtain activity data of the cell sample; in step 10), the palladium isotope signal intensity detection data includes signal intensity of at least one palladium isotope channel of 102 Pd、 104 Pd、 105 Pd、 106 Pd、 108 Pd and 110 Pd.
  2. 2. The method of claim 1, wherein the solution C is 1X phosphate buffer containing 2.5-5 mg/mL bovine serum albumin.
  3. 3. The method according to claim 1 or 2, wherein the solution P is 1X phosphate buffer.
  4. 4. The method according to claim 1 or 2, wherein the F solution is a phosphate buffer solution containing 1.6% by volume of paraformaldehyde.
  5. 5. The method according to claim 1 or 2, wherein the solution D has a final concentration of 125 nM 191/193 Ir and the solvent is a phosphate buffer containing 1.6% paraformaldehyde by volume.
  6. 6. The method according to claim 1, wherein the number of the cell samples is 200 to 300 ten thousand, and the volume of the diluted working solution added to the cells in the step 1) is 0.5 to 1ml; In the step 3), adding 2-4 mL of the solution C and the solution P into the palladium-activated stained cells obtained in the step 2); In the step 4), adding the F solution into the cells obtained by the treatment in the step 3) in an amount of 1-2 mL; in the step 5), the amount of the cells fixed in the step 4) added with the C solution and the P solution is 2-4 mL; in the step 6), the amount of the cells treated in the step 5) added into the M solution is 1-2 mL; in the step 7), the amount of the C solution and the P solution added into the cells subjected to the membrane rupture in the step 6) is 2-4 mL; in the step 8), the amount of the cell blown and added into the solution D after the treatment in the step 7) is 0.5-1 ml.
  7. 7. The method according to claim 1 or 6, wherein the centrifugation rate in each of steps 1), 3), 5), 7) is 300 to 500 x g, and the centrifugation time is 5 to 15 minutes; In the step 2), the incubation time is 3-10 minutes; in the step 4), the incubation time is 10-20 minutes; in the step 6), the incubation temperature is 4 ℃ and the incubation time is 20-30 minutes; in the step 8), the incubation time is 60-90 minutes; In the step 9), the rinsing frequency of the P liquid is 2-3 times, and the rinsing frequency of the double distilled water is 2-3 times.

Description

Cell sample activity detection kit and detection method based on mass spectrometry detection technology Technical Field The invention belongs to the technical field of mass spectrometry detection, and relates to a cell sample activity detection kit and a detection method based on mass spectrometry detection technology. Background Mass Cytometry (Mass Cytometry) is a high throughput single cell analysis technique combining flow Cytometry with Mass spectrometry techniques. By using metal isotope labeled antibodies or probes, mass flow cytometry is able to detect tens of different biomarkers simultaneously at the single cell level. Compared with the traditional fluorescence flow cytometry, the mass flow cytometry has higher multiplex detection capability and fewer signal overlapping problems, and shows remarkable advantages in deep analysis of complex samples. In flow assays, dead cells can nonspecifically adsorb antibodies, resulting in abnormal staining, causing significant detection errors, and even masking key biological signals. Therefore, the interference of dead cells can be effectively identified and eliminated, and the accuracy and the repeatability of detection data can be remarkably improved, so that the physiological state of the cell population in the sample can be reflected more truly. Currently, in the field of cell activity detection, traditional methods rely mainly on dyes or fluorescent labeling reagents, such as propidium iodide (Propidium Iodide, PI) and Annexin V/PI double staining, etc. Although these methods have been widely used in fluorescence flow platforms, their limitations are increasingly prominent, including problems of toxicity of the dye itself, severe overlap of signal spectra, interference with other labeling channels in multiplex assays, etc. More importantly, these dyes cannot be used in mass spectrometry platforms because they do not contain metal tags available for mass spectrometry detection, and it is difficult to meet the technical requirements of the platform for reagent metal labeling and signal resolution. To accommodate the detection requirements of mass spectrometry platforms, cisplatin (CISPLATIN) was introduced to differentiate between living and dead cells. The method utilizes the characteristic that cisplatin can penetrate dead cell membranes and form covalent bonds with intracellular proteins, thereby realizing the labeling of the cell activity state. However, the use of cisplatin still has a series of problems including complex preparation process, poor reagent stability, and certain toxicity to cells. In addition, at the moment of rapid development of mass spectrometry platforms, the existing activity detection methods have failed to meet the requirements of large-scale sample processing, standardized data generation and high-throughput research. The cell activity detection scheme has the characteristics of being specially optimized for a mass spectrum platform, having high adaptability, being simple and convenient in marking process, being suitable for an automatic flow, being high in reagent stability and toxicity, guaranteeing cell integrity, being clear in detection signal, high in sensitivity, supporting multichannel parallel reading, being good in commercialization potential and being convenient for wide deployment of clinic and scientific research. Therefore, the novel, stable, simple and convenient to operate and high in specificity cell activity detection kit and detection method are developed, and become important breaks for promoting the development and application of mass spectrometry flow type technology. Disclosure of Invention In order to solve the problems that in the prior art, living/dead cells are difficult to accurately distinguish and dead cell interference is effectively eliminated, and simultaneously improve the repeatability and expansibility of a detection technology and meet the adaptation requirements of multiple sample types and research targets, the invention provides a cell activity detection kit and a detection method based on a mass spectrometry detection technology. The method has good platform compatibility, easy operation and detection stability, can remarkably improve the accuracy and data quality of living cell identification in mass spectrometry detection, provides powerful technical support for the fields of immunology, oncology, stem cell research and the like, and has wide commercialization and clinical transformation prospects. The invention provides a method for distinguishing living cells from dead cells by mass spectrometry by utilizing a palladium isotope mixture labeling technology so as to improve the sensitivity and accuracy of detection. The invention aims to further optimize and verify a cell activity detection method based on a palladium isotope labeling technology, improves the sensitivity and accuracy of detection by improving the preparation and experimental flow of a kit, and expands the application of