CD34 + cell transport fluid and validity verification method thereof
By using human albumin, compound amino acids, glucose and PBS compositions as CD34+ cell transport fluid, the problem of decreased cell activity and high transportation cost when transporting CD34+ cells under conventional cold chain logistics conditions was solved, and efficient and economical cell transport effect was achieved.
Patent Information
- Application Number
- CN202510213328.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-26
- Publication Date
- 2025-05-30
AI Technical Summary
The existing CD34+ cell transport methods have problems such as decreased cell activity and high transportation costs during long-term transportation, especially under conventional cold chain logistics conditions of 4℃, it is difficult to ensure the vitality and stability of cells.
Human albumin, compound amino acids, glucose and phosphate buffered saline solution (PBS) compositions were used as CD34+ cell transport solution, and the optimal ratio was 1% human albumin, 0.5% compound amino acids and 0.1% glucose, and the balance was phosphate buffered saline solution (PBS).
This transport liquid can transport CD34+ cells for a long time under conventional cold chain logistics conditions at 4°C, ensuring that the cell viability is greater than 90% within 24 hours, and the cell phenotype is almost unchanged, reducing transportation costs.
Smart Images
Figure CN120052328A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell biology, and particularly to a CD34+ cell transport solution and a method for verifying its effectiveness. Background Art
[0002] Stem cells are primitive cells with self-renewal and multi-lineage differentiation potential. They are the origin cells of the body and the progenitor cells that form various tissues and organs of the human body. Hematopoietic stem cells, also known as pluripotent stem cells, are a group of primitive hematopoietic cells present in hematopoietic tissues. CD34+ is a clinical biomarker for hematopoietic stem cell transplantation (HSCT). Cells expressing CD34+ are the main source of the long-term regenerative potential of donor tissues in clinical hematopoietic stem cell transplantation. The surface expression of CD34+ protein usually gradually weakens as the cells mature and disappears when the cells mature into terminal effectors. Based on this expression pattern, CD34+ plays a core role in evaluating or selecting donor tissues in HSCT. CD34+ viable cell counting is clinically used to determine the cell dose obtained from the donor bone marrow or peripheral blood for hematopoietic stem cell transplantation (HSCT).
[0003] Currently, stem cell and immune cell technologies are widely used, and cell transportation has become an important link in the entire cell industry chain. There are two commonly used cell transportation methods. One is cryogenic storage and transportation, which is a transportation method using liquid nitrogen or dry ice in a special container. The preservation effect is good, but the disadvantage is that the transportation conditions are harsh, it is not suitable for long-term transportation, mostly requires air transportation, the transportation cost is relatively high, and the activity of the cryopreserved cells will decrease after resuscitation. The other is resuscitated cell transportation, which uses a cell culture flask filled with cell culture medium for transportation. This method has problems such as liquid leakage during long-distance transportation and the cell survival conditions and environment. Therefore, there is an urgent need for a transport solution that can meet the transportation of CD34+ cells under the conditions of 4°C conventional cold chain logistics. Summary of the Invention
[0004] The purpose of the present invention is to provide a CD34+ cell transport solution and a method for verifying its effectiveness to solve the problems raised in the above background art.
[0005] To achieve the above purpose, the present invention provides the following technical solution: A CD34+ cell transport solution, characterized in that the contents of each component are as follows by mass percentage: 1% - 5% human albumin, 0.5% - 1% compound amino acids, and 0.1% glucose, and the balance is phosphate buffered saline (PBS).
[0006] Preferably, the mass percentage contents of the respective components are: 5% human albumin, 0.5% compound amino acids, and 0.1% glucose, with the balance being phosphate buffered saline (PBS).
[0007] Preferably, the mass percentage contents of the respective components are: 1% human albumin, 1% compound amino acids, and 0.1% glucose, with the balance being phosphate buffered saline (PBS).
[0008] Preferably, the mass percentage contents of the respective components are: 1% human albumin, 0.5% compound amino acids, and 0.1% glucose, with the balance being phosphate buffered saline (PBS).
[0009] A method for validating the effectiveness of a CD34+ cell transport solution, characterized by comprising:
[0010] Step 1, obtaining CD34+ cells: taking scientific research umbilical cord blood and extracting CD34+ cells.
[0011] Step 2, establishing experimental groups: equally dividing the CD34+ cells extracted in Step 1 into three parts, and resuspending them to 1×10 6 / ml with transport solution A, transport solution B, and transport solution C respectively, and standing at 4°C to obtain the corresponding three experimental groups A, B, and C.
[0012] Step 3, detecting the experimental groups: for the three experimental groups established in Step 2, sampling every 6 hours, measuring the cell number and viability by trypan blue staining, and performing flow cytometry phenotype detection, monitoring at 0 hour, 6 hours, 18 hours, and 24 hours.
[0013] Step 4, obtaining the verification result: according to the cell viability and phenotype detection results in Step 3, it can be concluded that among the transport solutions used for CD34+ cells, transport solution C is superior to transport solution B, and transport solution B is superior to transport solution A.
[0014] Preferably, in Step 1, specifically: first pre-enriching umbilical cord blood CD34+ cells using a STEMCELL kit, and then using STEMCELLEasySep TM Human Umbilical Cord Blood CD34 Positive Selection Kit III to extract umbilical cord blood CD34+ cells. The specific operation steps for CD34+ enrichment and extraction are carried out according to the instructions of the EasySep TM Human Cord Blood CD34 Positive SelectionKit II (product number: 17896) kit.
[0015] Preferably, in the second step, the transport fluid A is PBS; the transport fluid B is 1% human albumin, and the balance is PBS; the transport fluid C is 1% human albumin, 0.5% compound amino acids, 0.1% glucose, and the balance is PBS.
[0016] Preferably, in the third step, the viability detection by trypan blue staining is specifically as follows: using an automatic cell counter for operation, mixing the cell suspension and 0.2% trypan blue solution evenly at a ratio of 1:1, and sucking 20 μL of the sample into a cleaned counting plate, inserting the counting plate into the cell analyzer and analyzing it to obtain the cell viability.
[0017] Preferably, in the third step, the phenotype detection by flow cytometry is specifically as follows:
[0018] 1) First, add reagents to 2 flow tubes respectively. Tube 1# contains 5 μL of CD45+ and 5 μL of IgG1PE, and tube 2# contains 5 μL of CD45+, 5 μL of CD34+ and 5 μL of 7 - AAD;
[0019] 2) Add 200 μL of cord blood CD34+ cell detection sample to tube 1# and tube 2# respectively, gently vortex and mix well, and incubate in the dark at room temperature for 15 min;
[0020] 3) Add 1.5 mL of PBS or sheath fluid to each flow tube respectively, gently shake and mix well, centrifuge at 500 g / min for 3 min, discard the supernatant after centrifugation, and use absorbent paper to suck the liquid remaining at the tube mouth. Add 200 μL of sheath fluid to each flow tube respectively, and gently shake to resuspend and mix well;
[0021] 4) According to the requirements of the "Standard Operating Procedure of Flow Cytometer (FACS Calibur)", execute the startup procedure and software connection, establish a sample collection template, use the software to call and collect samples, and obtain 10,000 cells for flow cytometry data analysis.
[0022] Preferably, the sample collection template is as follows: A Set the CD45+ gate: including all white blood cells and removing cell debris; B Display the cells within the CD45+ gate, set the 7 - AAD gate: including the 7 - AAD negative cell population; C Display the cells within the CD45+ gate, confirm the CD34+ gate according to the position of the negative cell mass of the isotype control (Mouse IgG1 PE); D Display the quantity - level percentage of each type of cell within each regional gate.
[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses a composition of human albumin, compound amino acids, glucose and phosphate buffered saline (PBS) as the CD34+ cell transport solution. Through experiments, the effectiveness and reliability of this transport solution have been verified. This transport solution can meet the long-term transportation of CD34+ cells under the conventional cold chain logistics conditions at 4°C, and can ensure that within 24 hours, the cell viability is greater than 90%, and the cell phenotype hardly changes. The present invention provides a new method for the transportation of CD34+ cells, effectively reducing the transportation cost and ensuring the cell viability. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 is the flowchart of the method of the present invention;
[0025] Figure 2 is the graph of the detection results of the cell viability of the three experimental groups in Example 1;
[0026] Figure 3 is the graph of the detection results of the CD34+ phenotype of the three experimental groups in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0027] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
[0028] Please refer to Figures 1 - 3 , a technical solution provided by the present invention:
[0029] Example 1:
[0030] A CD34+ cell transport solution, the formula includes: human albumin, compound amino acids, glucose and phosphate buffered saline (PBS). The mass percentage contents of each component are: 1% of human albumin, 0.5% of compound amino acids and 0.1% of glucose, and the balance is phosphate buffered saline (PBS).
[0031] A method for verifying the effectiveness of a CD34+ cell transport solution includes Step 1, obtaining CD34+ cells; Step 2, establishing experimental groups; Step 3, detecting the experimental groups; Step 4, obtaining verification results;
[0032] Among them, in the above Step 1, take scientific research umbilical cord blood and perform CD34+ cell extraction. Specifically: first pre-enrich umbilical cord blood CD34+ cells using the STEMCELL kit, and then use STEMCELL EasySep TMThe Human Umbilical Cord Blood CD34 Positive Selection Kit III was used to extract CD34+ cells from umbilical cord blood. The specific operation steps for CD34+ enrichment and extraction were carried out according to the instructions of the EasySep TM Human Cord Blood CD34 Positive Selection Kit II (product number: 17896);
[0033] In step 2 above, the CD34+ cells extracted in step 1 were evenly divided into three parts, and were resuspended to 1×10 6 / ml with Transport Solution A, Transport Solution B, and Transport Solution C respectively, and left to stand at 4°C to obtain the corresponding three experimental groups A, B, and C. Among them, Transport Solution A was PBS; Transport Solution B was 1% human albumin, and the remainder was PBS; Transport Solution C was 1% human albumin, 0.5% compound amino acids, 0.1% glucose, and the remainder was PBS;
[0034] In step 3 above, for the three experimental groups established in step 2, samples were taken every 6 hours, and trypan blue staining was used to measure the cell number and viability, and flow cytometry phenotype detection was carried out, monitoring 0 hours, 6 hours, 18 hours, and 24 hours; among them, the specific operation of trypan blue staining for viability detection was: using an automatic cell counter for operation, mixing the cell suspension and 0.2% trypan blue solution evenly at a ratio of 1:1, and sucking 20 μL of the sample into a washed counting plate, inserting the counting plate into a cell analyzer and analyzing it to obtain the cell viability; the specific operation of flow cytometry phenotype detection was:
[0035] 1) First, add reagents to 2 flow cytometry tubes. Tube 1# contains 5 μl of CD45+ and 5 μl of IgG1PE, and tube 2# contains 5 μl of CD45+, 5 μl of CD34+, and 5 μl of 7-AAD;
[0036] 2) Add 200 μl of umbilical cord blood CD34+ cell detection samples to tube 1# and tube 2# respectively, gently vortex and mix well, and incubate in the dark at room temperature for 15 min;
[0037] 3) Add 1.5 ml of PBS or sheath fluid to each flow cytometry tube respectively, gently shake and mix well, centrifuge at 500 g / min for 3 min, discard the supernatant after centrifugation, and use absorbent paper to suck the liquid remaining at the tube mouth. Add 200 μl of sheath fluid to each flow cytometry tube respectively, and gently shake and resuspend and mix well;
[0038] 4) Execute the startup procedure and software connection according to the requirements of the "Standard Operating Procedure for Flow Cytometer (FACS Calibur)", establish a sample collection template, use the software to call and collect samples, and obtain 10,000 cells for flow cytometry data analysis; the sample collection template is as follows: A Set the CD45+ gate: including all white blood cells and removing cell debris; B Display the cells within the CD45+ gate and set the 7-AAD gate: including the 7-AAD negative cell population; C Display the cells within the CD45+ gate and confirm the CD34+ gate according to the position of the negative cell mass of the isotype control (Mouse IgG1 PE); D Display the magnitude percentage of each type of cell within each regional gate.
[0039] Among them, in step 4 above, as Figure 2 , 3 shown, according to the cell viability and CD34+ phenotype detection results in step 3, it can be concluded that among the transport fluids used for CD34+ cells, transport fluid C is superior to transport fluid B, and transport fluid B is superior to transport fluid A.
[0040] Example 2:
[0041] A transport fluid for CD34+ cells, the formula includes: human albumin, compound amino acids, glucose and phosphate buffered saline (PBS), and the mass percentage contents of each component are: 5% human albumin, 0.5% compound amino acids and 0.1% glucose, and the balance is phosphate buffered saline (PBS).
[0042] A method for validating the effectiveness of a transport fluid for CD34+ cells, including step 1, obtaining CD34+ cells; step 2, establishing an experimental group; step 3, detecting the experimental group; step 4, obtaining the verification result;
[0043] Among them, in step 1 above, take scientific research umbilical cord blood and perform CD34+ cell extraction. Specifically: first pre-enrich umbilical cord blood CD34+ cells using the STEMCELL kit, and then use STEMCELL EasySep TM Human Cord Blood CD34 Positive Selection Kit III to extract umbilical cord blood CD34+ cells. The specific operation steps for CD34+ enrichment and extraction are carried out according to the instructions of the EasySep TM Human Cord Blood CD34 Positive Selection Kit II (product number: 17896) kit.
[0044] Among them, in step 2 above, divide the CD34+ cells extracted in step 1 into three equal parts, and resuspend them with transport fluid A, transport fluid B, and transport fluid C to 1×10 6 / ml, leave it standing at 4°C to obtain three corresponding experimental groups A, B, and C. Among them, the transport fluid A is PBS; the transport fluid B is 1% human serum albumin, and the balance is PBS; the transport fluid C is 5% human serum albumin, 0.5% compound amino acids, and 0.1% glucose, and the balance is phosphate buffered saline (PBS);
[0045] Among them, in the above step three, for the three experimental groups established in step two, samples are taken every 6 hours, and trypan blue staining is used to measure the cell number and viability, and flow cytometry phenotype detection is carried out, monitoring 0 hours, 6 hours, 18 hours, and 24 hours; among them, the viability detection by trypan blue staining method is specifically: use an automatic cell counter for operation, mix the cell suspension and 0.2% trypan blue solution evenly at a ratio of 1:1, and aspirate 20 μL of the sample into a cleaned counting plate, insert the counting plate into the cell analyzer and analyze it to obtain the cell viability; the flow cytometry phenotype detection is specifically:
[0046] 1) First, add reagents to 2 flow cytometry tubes. Tube 1# contains 5 μl of CD45+ and 5 μl of IgG1PE, and tube 2# contains 5 μl of CD45+, 5 μl of CD34+, and 5 μl of 7-AAD;
[0047] 2) Add 200 μl of cord blood CD34+ cell detection samples to tube 1# and tube 2# respectively, gently vortex and mix evenly, and incubate in the dark at room temperature for 15 min;
[0048] 3) Add 1.5 ml of PBS or sheath fluid to each flow cytometry tube respectively, gently shake and mix evenly, centrifuge at 500 g / min for 3 min, discard the supernatant after centrifugation, and use absorbent paper to absorb the liquid remaining at the tube mouth. Add 200 μl of sheath fluid to each flow cytometry tube respectively, and gently shake to resuspend and mix evenly;
[0049] 4) According to the requirements of the "Standard Operating Procedure of Flow Cytometer (FACS Calibur)", execute the startup procedure and software connection, establish a sample collection template, use the software to call and collect samples, and obtain 10,000 cells for flow cytometry data analysis; the sample collection template is as follows: A Set the CD45+ gate: include all white blood cells and remove cell debris; B Display the cells within the CD45+ gate, set the 7-AAD gate: include the 7-AAD negative cell population; C Display the cells within the CD45+ gate, and confirm the CD34+ gate according to the position of the isotype control (Mouse IgG1 PE) negative cell mass; D Display the quantity percentage of each type of cell within each regional gate.
[0050] Example 3:
[0051] A CD34+ cell transport solution, the formula includes: human serum albumin, compound amino acids, glucose and phosphate buffered saline (PBS). The mass percentage content of each component is: 1% human serum albumin, 1% compound amino acids and 0.1% glucose, and the balance is phosphate buffered saline (PBS).
[0052] A method for validating the effectiveness of a CD34+ cell transport solution, including Step 1, obtaining CD34+ cells; Step 2, establishing experimental groups; Step 3, detecting the experimental groups; Step 4, obtaining the verification results;
[0053] Among them, in the above Step 1, take scientific research umbilical cord blood and perform CD34+ cell extraction. Specifically: first pre-enrich umbilical cord blood CD34+ cells using the STEMCELL kit, and then use STEMCELLEasySep TM Human Cord Blood CD34 Positive Selection Kit III to extract umbilical cord blood CD34+ cells. The specific operation steps for CD34+ enrichment and extraction are carried out according to the instructions of the EasySep TM Human Cord Blood CD34 Positive Selection Kit II (product number: 17896) kit;
[0054] Among them, in the above Step 2, divide the CD34+ cells extracted in Step 1 into three equal parts, and resuspend them in Transport Solution A, Transport Solution B and Transport Solution C to 1×10 6 / ml, and let it stand at 4°C to obtain the corresponding three experimental groups A, B and C. Among them, Transport Solution A is PBS; Transport Solution B is 1% human serum albumin, and the balance is PBS; Transport Solution C is 1% human serum albumin, 1% compound amino acids and 0.1% glucose, and the balance is phosphate buffered saline (PBS);
[0055] Among them, in the above Step 3, for the three experimental groups established in Step 2, sample every 6 hours, use trypan blue staining to measure the cell number and viability, perform flow cytometry phenotype detection, and monitor 0 hours, 6 hours, 18 hours and 24 hours; among them, the viability detection by trypan blue staining method is specifically: use an automatic cell counter for operation, mix the cell suspension and 0.2% trypan blue solution evenly at a ratio of 1:1, and pipette 20 μL of the sample into a cleaned counting plate, insert the counting plate into the cell analyzer and analyze it to obtain the cell viability; the flow cytometry phenotype detection is specifically:
[0056] 1) First, add reagents to 2 flow cytometry tubes. Tube 1 is 5 μl CD45+ and 5 μl IgG1PE, and Tube 2 is 5 μl CD45+, 5 μl CD34+ and 5 μl 7-AAD;
[0057] 2) Add 200 μl of umbilical cord blood CD34+ cell detection samples to Tube 1# and Tube 2# respectively, gently vortex and mix well, and incubate in the dark at room temperature for 15 min;
[0058] 3) Add 1.5 ml of PBS or sheath fluid to each flow cytometry tube, gently shake and mix well, centrifuge at 500 g / min for 3 min, discard the supernatant after centrifugation, and use absorbent paper to absorb the residual liquid at the tube mouth. Add 200 μl of sheath fluid to each flow cytometry tube, and gently shake to resuspend and mix well;
[0059] 4) According to the requirements of the "Standard Operating Procedure of Flow Cytometer (FACS Calibur)", execute the startup procedure and software connection, establish a sample collection template, use the software to call and collect samples, and obtain 10,000 cells for flow cytometry data analysis; the sample collection template is as follows: A Set the CD45+ gate: including all white blood cells and removing cell debris; B Display the cells within the CD45+ gate, and set the 7-AAD gate: including the 7-AAD negative cell population; C Display the cells within the CD45+ gate, and confirm the CD34+ gate according to the position of the negative cell mass of the isotype control (Mouse IgG1 PE); D Display the quantitative percentage of each type of cell within each regional gate.
[0060] The property comparisons of each embodiment are as follows in the table:
[0061]
[0062] Based on the above, the present invention uses a composition of human albumin, compound amino acids, glucose and phosphate buffered saline (PBS) as the CD34+ cell transport solution, and determines its optimal ratio as 1% human albumin, 0.5% compound amino acids and 0.1% glucose, with the balance being phosphate buffered saline (PBS); this transport solution can meet the long-term transportation of CD34+ cells under the conventional cold chain logistics conditions at 4°C, and can ensure that within 24 hours, the cell viability is greater than 95%, and the cell phenotype hardly changes at all.
[0063] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.
Claims
1. A CD34+ cell transport fluid, characterized in that: The contents of the components in percentage by mass are: 1%-5% human albumin, 0.5%-1% compound amino acid and 0.1% glucose, and the remainder is phosphate buffered saline (PBS).
2. A CD34+ cell transport fluid according to claim 1, characterized in that: The mass percentage contents of the components are: 5% human albumin, 0.5% compound amino acids and 0.1% glucose, and the remainder is phosphate buffered saline (PBS).
3. A CD34+ cell transport fluid according to claim 1, characterized in that: The mass percentage contents of the components are: 1% human albumin, 1% compound amino acids and 0.1% glucose, and the remainder is phosphate buffered saline (PBS).
4. A CD34+ cell transport fluid according to claim 1, characterized in that: The mass percentage contents of the components are: 1% human albumin, 0.5% compound amino acids and 0.1% glucose, and the remainder is phosphate buffered saline (PBS).
5. A method for verifying the effectiveness of a CD34+ cell transport fluid, characterized in that: include: Step 1: Obtain CD34+ cells: Take umbilical cord blood for research and extract CD34+ cells; Step 2: Establish experimental groups: Divide the CD34+ cells extracted in step 1 into three equal parts and resuspend them in transport solution A, transport solution B, and transport solution C to 1×10 6 / ml, and placed at 4°C to obtain three corresponding experimental groups A, B and C; Step 3, testing the experimental groups: for the three experimental groups established in step 2, samples were taken every 6 hours, the cell number and viability were determined by trypan blue staining, and flow cytometry was performed to monitor 0 hours, 6 hours, 18 hours and 24 hours; Step 4, obtaining verification results: According to the cell viability and phenotype test results of step 3, it can be concluded that among the transport fluids used for CD34+ cells, transport fluid C is better than transport fluid B, and transport fluid B is better than transport fluid A.
6. The method for verifying the effectiveness of a CD34+ cell transport fluid according to claim 5, characterized in that: In step 1, specifically, the cord blood CD34+ cells were pre-enriched using the STEMCELL kit, and then the STEMCELL EasySep TM Human umbilical cord blood CD34 positive selection kit III is used to extract umbilical cord blood CD34+ cells.
7. The method for verifying the effectiveness of a CD34+ cell transport fluid according to claim 5, characterized in that: In step 2, transport liquid A is PBS; transport liquid B is 1% human albumin, and the balance is PBS; transport liquid C is 1% human albumin, 0.5% compound amino acids, 0.1% glucose, and the balance is PBS.
8. The method for verifying the effectiveness of a CD34+ cell transport fluid according to claim 5, characterized in that: In step 3, the trypan blue staining method is specifically used to detect the viability of The automatic cell counter was operated, and the cell suspension was mixed with 0.2% trypan blue solution in a 1:1 ratio. 20 μL of the sample was pipetted onto Wash the counting plate, insert the counting plate into the cell analyzer and analyze to obtain the cell viability.
9. The method for verifying the effectiveness of a CD34+ cell transport fluid according to claim 5, characterized in that: In step 3, the flow cytometer type detection is specifically as follows: 1) First, add reagents to two flow tubes respectively. Tube #1 contains 5 μl CD45+ and 5 μl IgG1PE, and tube #2 contains 5 μl CD45+, 5 μl CD34+, and 5 μl 7-AAD. 2) Add 200 μl of cord blood CD34+ cell test sample to tube 1# and tube 2# respectively, vortex gently to mix, and incubate at room temperature in the dark for 15 minutes; 3) Add 1.5 ml of PBS or sheath fluid to each flow tube, shake gently to mix, centrifuge at 500 g / min for 3 min, discard the supernatant after centrifugation, and absorb the residual liquid at the tube mouth with absorbent paper, add 200 μl of sheath fluid to each flow tube, shake gently to resuspend and mix; 4) According to the requirements of the "Flow Cytometer (FACS Calibur) Standard Operating Procedure", execute the startup procedure and software connection, establish a sample collection template, use the software to call and collect samples, and obtain 10,000 cells for flow data analysis.
10. The method for verifying the effectiveness of a CD34+ cell transport fluid according to claim 9, characterized in that: The sample collection template is as follows: A sets the CD45+ gate: includes all white blood cells and removes cell debris; B displays cells within the CD45+ gate and sets the 7-AAD gate: includes 7-AAD negative cell populations; C displays cells within the CD45+ gate and confirms the CD34+ gate based on the position of the isotype control (MouseIgG1PE) negative cell cluster; D displays the order of magnitude percentage of each type of cell within each area gate.