Anti-caking diluent for fluorescence imaging cell counter and use method of anti-caking diluent

Abstract

The invention relates to the technical field of fluorescence imaging cell counting, in particular to an anti-caking diluent for a fluorescence imaging cell counter and a use method of the anti-caking diluent, the anti-caking diluent comprises a buffer system, 1g / L inorganic salt, 2.0-4.0 mM metal ion chelating agent and 0.1-0.5% (w / v) surfactant, and the PH is 7.30 + / -0.1; according to the anti-caking diluent disclosed by the invention, through optimal design of core components, a key path of cell aggregation is specifically blocked from a microscopic level, and meanwhile, the cell activity is guaranteed. The nonionic surfactant can directly reduce the surface tension of cells, reduce the physical adsorption force between suspension cells and inhibit cell agglomeration from a physical mechanism; the low-concentration EDTA can specifically chelate Ca < 2 + > in the culture medium, and Ca < 2 + > is a key ion for mediating adhesion between cells, so that a biochemical pathway for cell adhesion can be blocked after chelation, and agglomeration is further prevented; the DPBS buffer system can stabilize the pH value of a dilution environment in a physiological range, and cell membrane damage or activity reduction caused by acid-base stress of cells is avoided.

Description

Technical Field

[0001] This invention relates to the field of fluorescence imaging cell counting technology, specifically to an anti-caking diluent for a fluorescence imaging cell counter and its method of use. Background Technology

[0002] In the fields of biopharmaceuticals (such as monoclonal antibody and vaccine production), cell therapy (such as CAR-T cell preparation), and clinical testing, cell counting and viability analysis are core steps in controlling product quality and ensuring experimental reliability. Among these, fluorescence imaging cell counters (such as Countess IIFL) have become the mainstream testing equipment in the industry due to their advantage of rapidly distinguishing between live and dead cells using AOPI dual-fluorescent dyes (acrididine orange AO / propidium iodide PI). The diluent, as a key auxiliary reagent in this testing process, is extremely critical, especially in meeting the requirements of "adjusting cells to the appropriate concentration, maintaining cell viability, preventing cell aggregation, and not interfering with the dye signal." Its performance directly determines the accuracy of the final test results.

[0003] Currently, the most widely used diluents in the industry are phosphate-buffered saline (PBS), physiological saline, and conventional cell culture media. However, these conventional reagents have significant technical drawbacks: First, they easily cause cell clumping. Suspended cells (such as CHO-K1 cells) tend to aggregate due to surface tension during dilution; adherent cells (such as HEK293 cells) also easily form clumps after trypsin digestion, exposing cell membrane adhesion sites. These clumps can lead to instrument misjudgment (counting multiple cell clusters as single cells or ignoring them altogether), severely affecting counting accuracy. Second, they have poor compatibility with AOPI dyes. Conventional diluents are not optimized for the dye's reaction environment, which may cause AO / PI dye molecules to depolymerize, fluorescence to be quenched, or promote non-specific binding of dyes to extracellular impurities, resulting in increased fluorescence background and overlapping fluorescence signals between live and dead cells, making accurate differentiation impossible. Third, they lack versatility. Most diluents are designed only for a specific type of suspended or adherent cell. When detecting different cell types, reagents need to be changed frequently, increasing experimental costs and reducing operational efficiency.

[0004] Therefore, the industry needs a dedicated diluent that can balance "low clumping, high compatibility, and wide applicability" to solve the core technical problems in the testing process. Summary of the Invention

[0005] This invention relates to an anti-caking diluent for a fluorescence imaging cell counter and its method of use.

[0006] This invention provides the following technical solution: An anti-caking diluent for a fluorescence imaging cell counter comprises: 1 g / L inorganic salt, 2.0-4.0 mM metal ion chelating agent, 0.1-0.5% (w / v) surfactant, buffer system balance, and pH 7.30±0.1; The buffer system is DPBS; The inorganic salt is NaCl; The metal ion chelating agent is EDTA; The surfactant is: Pluronic F-68; It is used in AOPI dyes.

[0007] As a further technical solution, the buffer system can also be replaced with HEPES or MOPS buffer.

[0008] As a further technical solution, the surfactant can also be replaced with polysorbates, such as Tween-20 and Tween-80, at a concentration of 0.005–0.05% (w / v); Or alkyl glycosides, such as cocoyl glucoside; Or a combination of multiple surfactants, such as Pluronic F-68+Tween-20.

[0009] As a further technical solution, the metal ion chelating agent can also be replaced with EGTA.

[0010] As a further technical solution, a cell protectant, such as 50-200mM sucrose or trehalose, can be added to the anti-caking diluent. Or antioxidants: such as glutathione and ascorbic acid, to prevent cellular oxidative stress; Alternatively, use a fluorescence quenching protectant, such as 2% (w / v) DABCO, to prevent dye photobleaching.

[0011] As a further technical solution, the diluent is not only compatible with AOPI dyes, but also applicable to other commonly used fluorescent dyes, such as trypan blue, for bright-field counting.

[0012] The method for using the anti-clumping diluent for a fluorescence imaging cell counter includes the following steps: a. Cell preparation: Take suspension cells in the logarithmic growth phase and adherent cells digested with trypsin, resuspend them in standard culture medium, and adjust the initial cell concentration to approximately 1×10⁻⁶. 6 cells / mL; b. Dilution treatment: The cell suspension was diluted 10-fold using the diluent described in this invention; a control group was set up using DPBS buffer for the same dilution. Control group: The cells were diluted using the same fractional dilution as the complete culture medium / DPBS used for cell resuspension. c. Staining treatment: Add AOPI dual fluorescent dye to the diluted cell suspension, mix well, and incubate at room temperature in the dark for 0–2 minutes; d. Imaging and counting: Take an appropriate amount of stained sample and load it into the counting chamber of the imaging cell counter; start the instrument to perform automatic focusing and fluorescence imaging to acquire bright field and fluorescence images; Specifically, take 10 μL of cell suspension and 10 μL of AOPI dye, mix them thoroughly, add them to a cell counting chamber, and then count the cells. e. Data Analysis: The instrument software automatically identifies and statistically analyzes: total cell count; viable cell count (AO positive); dead cell count (PI positive); cell clumping rate; f. Fluorescence background intensity: Tested using a TECAN multi-mode microplate reader. Diluent: AOPI non-specific fluorescence interference.

[0013] As a further technical solution, the suspended cells in step a include CHO-K1 and lymphoid progenitor cells.

[0014] As a further technical solution, the adherent cells digested by trypsin in step a include HEK293 and RAW264.7 cells.

[0015] As a further technical solution, in step a, the conventional culture medium refers to the complete culture medium corresponding to the cell. Resuspension method: Add an appropriate amount of complete culture medium; Gently pipette 5-10 times until the cells are completely dispersed and there are no obvious clumps.

[0016] Compared with the prior art, the beneficial effects of the present invention are: The anti-agglomeration diluent of this invention, through optimized design of its core components, specifically blocks key pathways of cell aggregation at the microscopic level while ensuring cell viability. Specifically, nonionic surfactants (such as Pluronic F-68) directly reduce cell surface tension, decreasing the physical adsorption forces between suspended cells and inhibiting cell aggregation through a physical mechanism; low-concentration EDTA specifically chelates Ca in the culture medium. 2+ And Ca 2+It is a key ion mediating intercellular adhesion; after chelation, it can block the biochemical pathways of cell adhesion, further preventing clumping. The DPBS buffer system can stabilize the pH of the dilution environment within the physiological range (7.2-7.4), avoiding cell membrane damage or decreased activity due to acid-base stress. By targeting the three major problems of cell clumping—physical adsorption, biochemical adhesion, and environmental stress—these components can achieve anti-clumping and cell protection effects, effectively solving the problem that existing diluents only have basic buffering functions and lack targeted anti-clumping design, leading to counting errors.

[0017] The synergistic effect of the components in this invention further overcomes the contradiction between anti-caking and dye compatibility and cell adaptation, achieving a performance balance at the meso-level. On the one hand, the nonionic surfactant (such as Pluronic F-68) used in this invention is different from the traditional Triton X-100 (which easily damages the dye structure). Its chemical properties are mild and have no effect on the stability of AOPI dye molecules. At the same time, EDTA is controlled at a low concentration to avoid damage to the cell membrane by high-concentration chelating agents. Combined with the optimized ionic strength of the DPBS system, the non-specific binding of dye to extracellular impurities can be reduced. This synergistic design of surfactant type selection, chelating agent concentration control, and buffer system ion optimization results in clear and stable green (AO) fluorescence signals in live cells and red (PI) fluorescence signals in dead cells after AOPI staining, with a significant reduction in fluorescence background interference. This solves the problem that existing diluents are prone to anti-caking but destroying the dye effect or being compatible with the dye but unable to prevent clumping. On the other hand, the cell compatibility of the surfactant complements the environmental stability of the buffer system, making the diluent suitable for both suspension cells such as CHO-K1 and lymphocyte progenitor cells, and adherent cells such as HEK293 and RAW264.7 that have been digested with trypsin. This eliminates the need to change reagents for different cell types and effectively solves the problem of low experimental efficiency caused by the lack of versatility of existing diluents.

[0018] From the overall macroscopic effect of the solution, the performance advantages of the diluent in high-dilution scenarios can meet the core needs of the industry and create additional value. Under 10-fold dilution (a common multiple for high-density cell detection in biopharmaceuticals), thanks to the synergistic effect of the aforementioned components, the agglomeration rate of both suspended and adherent cells decreased by more than 5%, the uniformity of cell distribution was greatly improved, and the accuracy of instrument image recognition was significantly improved. At the same time, the total cell count was highly consistent with the theoretical dilution value, with no counting deviation caused by cell loss or aggregation, and the cell viability was not significantly different from the control group (DPBS) (p>0.05), proving that the diluent, while ensuring counting accuracy, does not affect cell viability or the accuracy of AOPI dye in recognizing live and dead cells. This comprehensive performance enables it to meet the detection needs of high-density cultured cells in biopharmaceuticals (such as high-concentration cells in CAR-T production), and can replace imported diluents such as ThermoFisher and Merck, reducing the industry's dependence on imported reagents. In addition, this diluent can also be used with imaging cell counters and extended to scenarios such as pretreatment of flow cytometry and preparation of single-cell sequencing samples, thereby enabling the construction of a closed loop of standardized cell detection processes for domestically produced equipment and consumables, and promoting the independent development of domestic high-end biological reagents. Attached Figure Description

[0019] Figure 1 Bright field plot of CHO cell suspension; Figure 2 Bright-field plot of CHO cells diluted 10-fold with DPBS; Figure 3 Bright field plot of CHO cell dilution 10-fold; Figure 4 Bright field plot of CHO cell type 2 diluted 10-fold; Figure 5 Bright field plot of CHO cells diluted 10-fold with dilution 3; Figure 6 Bright field plot of CHO cells diluted 10-fold with dilution 4; Figure 7 Bright field plot of HEK293 cell suspension; Figure 8 Bright-field plot of HEK293 cells diluted 10-fold with DPBS; Figure 9 Bright-field plot of HEK293 cells diluted 10-fold with dilution 1; Figure 10 Bright-field plot of HEK293 cells diluted 10-fold with dilution 2; Figure 11 Bright-field plot of HEK293 cells diluted 10-fold with dilution 3; Figure 12Bright-field plot of HEK293 cells diluted 10-fold with dilution 4. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] An anti-caking diluent for a fluorescence imaging cell counter comprises: 1 g / L inorganic salt, 2.0-4.0 mM metal ion chelating agent, 0.1-0.5% (w / v) surfactant, buffer system balance, and pH 7.30±0.1; The buffer system is DPBS; The inorganic salt is NaCl; The metal ion chelating agent is EDTA; The surfactant is: Pluronic F-68; It is used in AOPI dyes.

[0022] In the anti-agglomeration diluent, DPBS serves as the base buffer, while surfactants and metal ion chelators act as anti-agglomeration agents.

[0023] The buffer system can also be replaced with HEPES or MOPS buffer.

[0024] Surfactants can also be replaced with polysorbates, such as Tween-20 and Tween-80, at a concentration of 0.005–0.05% (w / v); Or alkyl glycosides, such as cocoyl glucoside; Or a combination of multiple surfactants, such as Pluronic F-68+Tween-20.

[0025] Metal ion chelating agents can also be replaced with EGTA.

[0026] Cell protectants, such as 50-200mM sucrose or trehalose, can also be added to the anti-caking diluent. Or antioxidants: such as glutathione and ascorbic acid, to prevent cellular oxidative stress; Alternatively, use a fluorescence quenching protectant, such as 2% (w / v) DABCO, to prevent dye photobleaching.

[0027] The diluent is not only compatible with AOPI dyes, but also suitable for other commonly used fluorescent dyes, such as trypan blue, for bright-field counting.

[0028] The method for using the anti-clumping diluent for a fluorescence imaging cell counter includes the following steps: a. Cell preparation: Take suspension cells in the logarithmic growth phase and adherent cells digested with trypsin, resuspend them in standard culture medium, and adjust the initial cell concentration to approximately 1×10⁻⁶. 6 cells / mL; Suspended cells include CHO-K1 and lymphoid progenitor cells.

[0029] Adherent cells digested with trypsin included HEK293 and RAW264.7 cells.

[0030] Standard culture medium: refers to the complete culture medium corresponding to this cell; Resuspension method: Add an appropriate amount of complete culture medium; Gently pipette 5-10 times until the cells are completely dispersed and there are no obvious clumps.

[0031] b. Dilution treatment: The cell suspension was diluted 10-fold using the diluent described in this invention; a control group was set up using DPBS buffer for the same dilution. Control group: The cells were diluted using the same fractional dilution as the complete culture medium / DPBS used for cell resuspension. c. Staining treatment: Add AOPI dual fluorescent dye to the diluted cell suspension, mix well, and incubate at room temperature in the dark for 0–2 minutes; d. Imaging and counting: Take an appropriate amount of stained sample and load it into the counting chamber of the imaging cell counter; start the instrument to perform automatic focusing and fluorescence imaging to acquire bright field and fluorescence images; Specifically, take 10 μL of cell suspension and 10 μL of AOPI dye, mix them thoroughly, add them to a cell counting chamber, and then count the cells. e. Data Analysis: The instrument software automatically identifies and statistically analyzes: total cell count; viable cell count (AO positive); dead cell count (PI positive); cell clumping rate; f. Fluorescence background intensity: Tested using a TECAN multi-mode microplate reader. Diluent: AOPI non-specific fluorescence interference.

[0032] The following are examples of specific diluents, including diluents 1-4: Table 1 Buffer screening: Objective: To compare the pH stability of the two buffer solutions; Test method: Test at room temperature for 24 hours. Test quantity: 3 parallel groups; Table 2 Conclusion: The ΔpH of both buffer solutions is within acceptable limits.

[0033] Objective: To compare the fluorescence interference rates of the two buffer solutions. Test method: Compare the fluorescence intensity ratio of AOPI fluorescent dye to substrate solution; Test quantity: 3 parallel groups; Table 3 Conclusion: The fluorescence signal-to-noise ratios of both buffer solutions were satisfactory.

[0034] Objective: To compare the effects of two buffer solutions on cell viability; Test method: Compare the viability of T cells after dilution; Test quantity: 3 parallel groups; Table 4 Conclusion: DPBS is acceptable when diluted 10 times.

[0035] Anti-aggregating agent screening: Objective: To test the effects of EDTA anti-aggregation agent on T cell viability and aggregation rate; Test method: Compare the changes in viability and clumping rate of T cells before and after incubation for 0 min and 2 min, cell density: 2.07E6 / ml; Test quantity: 5 parallel groups; Table 5 Conclusion: The acceptable concentration of EDTA for activity and anti-caking is 2-5 mM.

[0036] Validation objective: To test the effect of EDTA anti-aggregation agent on CHO cell viability and aggregation rate; Test method: Compare the changes in viability and clumping rate of CHO cells before and after incubation for 0 min and 2 min, cell density: 4.14E6 / ml; Test quantity: 5 parallel groups; Table 6 Conclusion: The acceptable concentrations for EDTA in terms of activity and anti-caking are 2 mM and 4 mM, with 4 mM being the optimal concentration.

[0037] Validation objective: To test the effects of Pluronic F-68 on cell viability and clumping rate; Test method: Compare the changes in viability and clumping rate of CHO cells before and after incubation for 0 min and 2 min; Test quantity: 5 parallel groups; Table 7 Conclusion: The optimal concentration of Pluronic F-68 for achieving satisfactory survival and anti-caking properties is 0.10%.

[0038] Without adding inorganic salts, perform the following preliminary dilution tests 1-4: Diluent 1: DPBS + EDTA (2mM) + Pluronic F-68 (0.1%); Diluent 2: DPBS + EDTA (2mM) + Pluronic F-68 (0.5%); Diluent 3: DPBS + EDTA (4mM) + Pluronic F-68 (0.1%); Diluent 4: DPBS + EDTA (4mM) + Pluronic F-68 (0.5%); Fluorescence background test: Table 8 Conclusion: The fluorescence background of all four diluents was within acceptable limits.

[0039] Validation objective: To test the effect of initial cell dilutions 1-4 on cell viability and clumping rate; Test method: Compare the changes in viability and clumping rate of CHO cells before and after 2 min of incubation; cell density: 1.62E6 / ml; Test quantity: 5 parallel groups; Table 9 Conclusion: Diluents 3 and 4 are qualified.

[0040] Verification objective: To test whether the freezing point osmotic pressure of the initial cell dilution buffer (1-4) conforms to the cell osmotic pressure range; Test method: The freezing point osmotic pressure of cell diluent V1.0 was tested using a freezing point osmometer; Test quantity: 3 parallel groups; Table 10 Conclusion: The osmotic pressure of diluents 2, 3 and 4 is too low, and inorganic salt (NaCl) needs to be added to increase the osmotic pressure.

[0041] Inorganic salts were added to obtain the optimized diluent, which is the final diluent 1-4 used in this invention: Diluent 1: DPBS + EDTA (2mM) + F-68 (0.1%) + NaCl (1g / L); Diluent 2: DPBS + EDTA (2mM) + F-68 (0.5%) + NaCl (1g / L); Diluent 3: DPBS + EDTA (4mM) + F-68 (0.1%) + NaCl (1g / L); Diluent 4: DPBS + EDTA (4mM) + F-68 (0.5%) + NaCl (1g / L); Validation objective: To test the freezing point osmotic pressure of cell dilution buffer 1-4; Test method: Use a freezing point osmometer to test the freezing point osmotic pressure of cell diluent 1-4; Test quantity: 3 parallel groups; Table 11 Conclusion: The osmotic pressure of diluents 1, 2, 3 and 4 is within acceptable limits.

[0042] Validation objective: To test the fluorescence interference rate of cell dilution buffers 1-4; Test method: Compare the fluorescence ratio of the diluted solution and the AOPI dye; Test quantity: 3 parallel groups; Table 12 Conclusion: The fluorescence background of diluents 1, 2, 3 and 4 is acceptable.

[0043] Objective: To test the effects of cell dilutions 1-4 on the viability and clumping rate of suspended cells; Test method: Compare the changes in viability and clumping rate of CHO cells before and after 2 min of incubation; cell density: 1.6E6 / ml; Test quantity: 5 parallel groups; Table 13 Conclusion: Diluents 2, 3, and 4 are qualified. Figures 1-6 It can be seen that the cell diluent affects the viability and clumping rate of suspended cells (CHO); Figure 1 Bright field plot of CHO cell suspension; Figure 2 Bright-field plot of CHO cells diluted 10-fold with DPBS; Figure 3 Bright field plot of CHO cell dilution 10-fold; Figure 4 Bright field plot of CHO cell type 2 diluted 10-fold; Figure 5 Bright field plot of CHO cells diluted 10-fold with dilution 3; Figure 6 Bright field plot of CHO cell dilution 4 diluted 10-fold.

[0044] Objective: To test the effects of cell dilutions 1-4 on the viability and clumping rate of suspended cells; Test method: Compare the changes in viability and clumping rate of lymphocyte progenitor cells before and after 2 min of incubation; cell density: 1.5E6 / ml; Test quantity: 5 parallel groups; Table 14 Conclusion: Diluents 1, 2, and 4 are qualified.

[0045] Objective: To test the effects of cell dilutions 1-4 on adherent cell viability and clumping rate; Test method: Compare the changes in viability and clumping rate of RAW264.7 adherent cells before and after incubation for 2 min, cell density: 1.64E6 / ml; Test quantity: 5 parallel groups; Table 15 Conclusion: Diluents 3 and 4 are qualified.

[0046] Objective: To test the effects of cell dilutions 1-4 on adherent cell viability and clumping rate; Test method: Compare the changes in viability and clumping rate of HEK293 adherent cells before and after 2 min of incubation; cell density: 2.0E6 / ml; Test quantity: 5 parallel groups; Table 16 Conclusion: Diluent No. 3 is qualified. Figures 7-12 The effects of cell dilution on the viability and clumping rate of adherent cells (HEK293) can be observed. Figure 7 Bright field plot of HEK293 cell suspension; Figure 8 Bright-field plot of HEK293 cells diluted 10-fold with DPBS; Figure 9 Bright-field plot of HEK293 cells diluted 10-fold with dilution 1; Figure 10 Bright-field plot of HEK293 cells diluted 10-fold with dilution 2; Figure 11 Bright-field plot of HEK293 cells diluted 10-fold with dilution 3; Figure 12 Bright-field plot of HEK293 cells diluted 10-fold with dilution 4.

[0047] Validation objective: To test the effects of cell dilutions 1-4 on the viability and clumping rate of highly adhesive cells; Test method: Compare the changes in viability and clumping rate of Jurkat cells before and after 2 min of incubation; cell density: >1E6 / ml; Test quantity: 5 parallel groups; Table 17 Conclusion: All four diluents were substandard.

[0048] The cell dilution buffer test results are summarized and compared below: Table 18 In summary, the final dilution 3 is suitable for AO / PI viability assays of a variety of common suspension and adherent cells (except Jurkat cells). It has no significant effect on cell viability within a 10-fold dilution range, while effectively reducing cell aggregation and improving cell dispersion and counting accuracy.

[0049] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not describe all details exhaustively, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification.

Claims

1. An anti-caking diluent for use in a fluorescence imaging cell counter, characterized in that, include: 1 g / L inorganic salt, 2.0-4.0 mM metal ion chelating agent, 0.1-0.5% (w / v) surfactant, buffer balance, pH 7.30±0.1; The buffer system is DPBS; The inorganic salt is NaCl; The metal ion chelating agent is EDTA; The surfactant is: Pluronic F-68; It is used in AOPI dyes.

2. The anti-caking diluent for a fluorescence imaging cell counter as described in claim 1, characterized in that, The buffer system can also be replaced with HEPES or MOPS buffer.

3. The anti-caking diluent for a fluorescence imaging cell counter as described in claim 1, characterized in that, The surfactant can also be replaced with polysorbates, such as Tween-20 and Tween-80, at a concentration of 0.005–0.05% (w / v); Or alkyl glycosides, such as cocoyl glucoside; Or a combination of multiple surfactants, such as Pluronic F-68+Tween-20.

4. The anti-caking diluent for a fluorescence imaging cell counter as described in claim 1, characterized in that, The metal ion chelating agent can also be replaced with EGTA.

5. The anti-caking diluent for a fluorescence imaging cell counter as described in claim 1, characterized in that, The anti-caking diluent may also contain cell protectants, such as 50-200 mM sucrose or trehalose. Or antioxidants: such as glutathione, ascorbic acid; Alternatively, a fluorescence quenching protectant, such as 2% (w / v) DABCO.

6. The anti-caking diluent for a fluorescence imaging cell counter as described in claim 1, characterized in that, The diluent is not only compatible with AOPI dyes, but also suitable for other commonly used fluorescent dyes, such as trypan blue, for bright-field counting.

7. A method of using the anti-caking diluent for a fluorescence imaging cell counter as described in any one of claims 1-6, characterized in that, Includes the following steps: a. Cell preparation: Take suspension cells in the logarithmic growth phase and adherent cells digested with trypsin, resuspend them in standard culture medium, and adjust the initial cell concentration to approximately 1×10⁻⁶. 6 cells / mL; b. Dilution treatment: The cell suspension was diluted 10-fold using the diluent described in this invention; a control group was set up using DPBS buffer for the same dilution. Control group: The cells were diluted using the same fractional dilution as the complete culture medium / DPBS used for cell resuspension. c. Staining treatment: Add AOPI dual fluorescent dye to the diluted cell suspension, mix well, and incubate at room temperature in the dark for 0–2 minutes; d. Imaging and counting: Take an appropriate amount of stained sample and load it into the counting chamber of the imaging cell counter; start the instrument to perform automatic focusing and fluorescence imaging to acquire bright field and fluorescence images; Specifically, take 10 μL of cell suspension and 10 μL of AOPI dye, mix them thoroughly, add them to a cell counting chamber, and then count the cells. e. Data Analysis: The instrument software automatically identifies and statistically analyzes: total cell count; viable cell count (AO positive); dead cell count (PI positive); cell clumping rate; f. Fluorescence background intensity: Tested using a TECAN multi-mode microplate reader. Diluent: AOPI non-specific fluorescence interference.

8. The method of using the anti-caking diluent for a fluorescence imaging cell counter as described in claim 7, characterized in that, The suspended cells in step a include CHO-K1 cells and lymphoid progenitor cells.

9. A method for using an anti-caking diluent for a fluorescence imaging cell counter as described in claim 6, characterized in that, The adherent cells digested by trypsin in step a include HEK293 and RAW264.7 cells.

10. A method for using an anti-caking diluent for a fluorescence imaging cell counter as described in claim 6, characterized in that, In step a, the conventional culture medium refers to the complete culture medium corresponding to that cell. Resuspension method: Add an appropriate amount of complete culture medium; Gently pipette 5-10 times until the cells are completely dispersed and there are no obvious clumps.

Images