Cell cleaning fluid and application thereof

Abstract

The invention discloses a cell cleaning fluid and application thereof, and belongs to the technical field of biology, the cell cleaning fluid comprises a PBS solution, glucose, EDTA and collagen; furthermore, the invention also discloses a method for preparing a cell quality control product by adopting the cell cleaning solution. According to the cleaning solution, a PBS buffer solution is used as a substrate to provide a basic buffer system, collagen provides protection for cells, and it is guaranteed that forms of cell membranes and components in the cells are complete; glucose and collagen act together to change the density and viscosity of the buffer solution, and the capacity of removing impurities in cells of the cleaning solution is improved; the surfactant EDTA increases the dispersibility of the cell quality control product and ensures the final IHC detection dyeing effect; and finally, an ideal effect is achieved in the aspects of cleaning and dispersing.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a cell cleaning solution and application thereof. Background Art

[0002] In the fields of medicine, biology, life sciences, etc., cell suspensions extracted from tissues, organs, culture fluids and other cell sampling sites usually contain impurities other than cells, which may have an adverse effect on research and analysis. Cell quality control products are a type of cell control product used to monitor the IHC detection process. Some cells are prone to bring in impurities during the preparation of quality control products, which affect the staining results of IHC detection. Therefore, it is necessary to remove these impurities through cell washing to improve the reliability of research and analysis results; cell washing is an important task. However, the PBS buffer conventionally used for cell washing cannot effectively remove staining impurities, resulting in poor washing effect. Summary of the invention

[0003] In order to solve at least one of the above problems, the present invention provides a cell cleaning solution and application thereof.

[0004] In order to achieve the above object, the present invention adopts the following technical means: A first aspect of the present invention provides a cell washing solution comprising PBS solution, glucose, EDTA and collagen.

[0005] In some embodiments of the present invention, the concentration of the PBS is 8-10 mM; preferably, the concentration of the PBS is 10 mM.

[0006] In some embodiments of the present invention, the mass volume percentage concentration of the glucose is 1%-7%; preferably, the mass volume percentage concentration of the glucose is 4%.

[0007] In some embodiments of the present invention, the mass volume percentage concentration of the EDTA is 0.01%-0.07%. Preferably, the mass volume percentage concentration of the EDTA is 0.04%.

[0008] In some embodiments of the present invention, the mass volume percentage concentration of the collagen is 0.005%-0.15%. Preferably, the mass volume percentage concentration of the collagen is 0.01%.

[0009] The second aspect of the present invention provides use of the cell washing solution described in the first aspect in preparing a washing solution for cell immunohistochemistry quality control products.

[0010] The third aspect of the present invention provides a method for preparing a cell quality control product, comprising the following steps: (1) preparing the cell washing solution described in the first aspect; (2) Collect the target cells, resuspend them in cell washing solution, and then centrifuge and wash them 1-2 times; (3) The washed cells are fixed, dehydrated, embedded, and sliced ​​to prepare a cell semi-solid suspension to obtain a cell quality control product. The preparation of the cell semi-solid suspension refers to the relevant method in patent CN 202011356471.8.

[0011] In some embodiments of the present invention, the centrifugation condition is 800-1000 RPM, preferably, the centrifugation condition is 900 RPM.

[0012] The fourth aspect of the present invention provides a cell quality control product prepared by the method described in the third aspect.

[0013] The fifth aspect of the present invention provides a cell immunohistochemistry detection kit, comprising the cell washing solution described in the first aspect.

[0014] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a cell washing solution, which uses PBS buffer as a base to provide a basic buffer system, and collagen provides protection for cells to ensure the integrity of the cell membrane and the morphology of each component in the cell; glucose and collagen work together to change the density and viscosity of the buffer, thereby increasing the ability of the washing solution to remove impurities in the cells; the surfactant EDTA increases the dispersibility of cell quality control products, thereby ensuring the final IHC detection staining effect; ultimately, ideal effects are achieved in terms of both washing and dispersion, and the cell washing solution can be applied to the washing of cell quality control products, and the cell washing solution has the same effect of washing and removing impurities for different cell lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure shows the dyeing effect of 5 groups of experiments in Example 1 of the present invention; Figure 2 The figure shows the dyeing effect of 4 groups of experiments in Example 2 of the present invention; Figure 3 The figures show the dyeing effects of three groups of experiments in Example 3 of the present invention; Figure 4 The figures show the dyeing effects of two groups of experiments in Example 4 of the present invention. DETAILED DESCRIPTION

[0016] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0018] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0019] Example 1 1. Resuscitate a strain of MCF7 cells (human breast cancer cells, Guangzhou Saiku CC0302) and expand the culture.

[0020] 2. Collect 100M cells in the logarithmic growth phase with a cell viability of 97.3%; 20M cells are grouped into 5 groups: Group 1, Group 2, Group 3, Group 4 and Group 5.

[0021] The composition of the cleaning solution is shown in Table 1 below: Table 1 Composition of cleaning solution

[0022] Note: The concentrations of glucose, EDTA and collagen in the table are in percentage by mass and volume.

[0023] The cells of group 2 to group 5 were resuspended to 40 mL using different component washing solution formulas, and washed once by centrifugation at 900 RPM.

[0024] 3. After collecting cells from group 1, resuspend them in 10mL 10mM PBS; prepare a 50mL centrifuge tube, add 10mL cell separation solution (human peripheral blood lymphocyte separation solution, Tianjin Haoyang LTS10771), and then slowly add 10mL of cell suspension along the side wall of the centrifuge tube onto the cell separation solution (move slowly so as not to damage the separation solution layer). Put the centrifuge tube to be separated into a centrifuge, set it to 1000RPM for 30 minutes, set the centrifugal acceleration to 5, and set the centrifugal deceleration to 0. Use a pipette to absorb the cell layer between PBS and the separation solution of the separated cell suspension, add it to 40mL 10mM PBS, mix well, and centrifuge at 900RPM to wash once.

[0025] 4. After washing, the cells in Groups 1 to 5 were fixed with 10% neutral formalin fixative for 2 hours and then washed to remove the fixative.

[0026] 5. After fixation, the cells were dehydrated using 50%, 75%, 95% and anhydrous ethanol for 1 hour respectively and then immersed in xylene for 30 minutes for clarity.

[0027] 6. After clearing, the cells were immersed in paraffin for 3 hours and embedded into blocks.

[0028] 7. The wax blocks were sliced ​​separately and prepared into cell suspensions according to patent CN 202011356471.8 A method for preparing a semi-solid cell suspension and its application. The cell suspension was prepared for immunohistochemical staining experiments; the immunohistochemical staining experiment was carried out using the LYNX480 fully automatic immunohistochemical staining machine of Tuling (Hangzhou) Biotechnology Co., Ltd. The target primary antibody (I1219) and auxiliary reagents (I20032C) used in conjunction were purchased from the commercial kit of Tuling (Hangzhou) Biotechnology Co., Ltd. The experiment adopted the conventional immunohistochemical experimental process and was carried out according to the instructions of the kit.

[0029] 8. Experimental results are as follows Figure 1 shown.

[0030] The results showed that the commercial cell separation solution was not effective in removing impurities from the quality control cells, and there was basically no improvement in impurity removal; when only PBS was used for removal, cells aggregated and large impurities adhered; when sugars were added alone to the 10mM PBS solution, there was basically no improvement in impurity removal; when EDTA was added alone, the cell dispersion increased, but there was basically no improvement in impurity removal; when collagen was added alone, the cells were not significantly dispersed, and there was basically no effect on impurity removal. It can be seen that the addition of EDTA has a significant effect on the degree of cell dispersion.

[0031] Example 2 1. Resuscitate a strain of MCF7 cells (human breast cancer cells, Guangzhou Saiku CC0302) and expand the culture.

[0032] 2. Collect 80M cells in the logarithmic growth phase with a cell viability of 96.7%; 20M cells were grouped into 4 groups: Group 6, Group 7, Group 8, and Group 9.

[0033] 3. The cells of groups 6 to 9 were resuspended to 40 mL using different washing solution formulas and washed once by centrifugation at 900 RPM. The composition of the washing solution is shown in Table 2 below: Table 2 Composition of cleaning fluid

[0034] Note: The concentrations of glucose, EDTA and collagen in the table are in percentage by mass and volume.

[0035] 4. After washing, the cells in groups 6 to 9 were fixed with 10% neutral formalin fixative for 2 hours and then washed to remove the fixative.

[0036] 5. After fixation, the cells were dehydrated using 50%, 75%, 95% and anhydrous ethanol for 1 hour respectively and then immersed in xylene for 30 minutes for clarity.

[0037] 6. After clearing, the cells were immersed in paraffin for 3 hours and embedded into blocks.

[0038] 7. The wax blocks were sliced ​​separately and prepared into cell suspensions according to patent CN 202011356471.8 A method for preparing a semi-solid cell suspension and its application. The cell suspension was prepared for immunohistochemical staining experiments; the immunohistochemical staining experiment was carried out using the LYNX480 fully automatic immunohistochemical staining machine of Tuling (Hangzhou) Biotechnology Co., Ltd. The target primary antibody (I1219) and auxiliary reagents (I20032C) used in conjunction were purchased from the commercial kit of Tuling (Hangzhou) Biotechnology Co., Ltd. The experiment adopted the conventional immunohistochemical experimental process and was carried out according to the instructions of the kit.

[0039] 8. Experimental results are as follows Figure 2 shown.

[0040] The results showed that adding glucose and EDTA to 10mM PBS solution could enhance the effect of the cleaning solution, increase cell dispersion and reduce impurities; adding glucose and collagen caused cell clustering and reduced impurities; adding EDTA and collagen dispersed the cells and there was basically no improvement in impurity removal; adding glucose, EDTA and collagen at the same time dispersed the cells and significantly removed impurities, achieving a better cleaning effect.

[0041] Example 3 1. Resuscitate a strain of MCF7 cells (human breast cancer cells, Guangzhou Saiku CC0302) and expand the culture.

[0042] 2. Collect 60M cells in the logarithmic growth phase with a cell viability of 96.9%; 20M cells were grouped into 3 groups: Group 10, Group 11, and Group 12.

[0043] 3. The cells of groups 10 to 12 were resuspended to 40 mL using washing solution formulas with different concentration components, and washed once by centrifugation at 900 RPM. The composition of the washing solution is shown in Table 3 below: Table 3 Composition of cleaning solution

[0044] Note: The concentrations of glucose, EDTA and collagen in the table are in percentage by mass and volume.

[0045] 4. The washed cells were fixed with 10% neutral formalin fixative for 2 hours, and then washed to remove the fixative.

[0046] 5. After fixation, the cells were dehydrated using 50%, 75%, 95% and anhydrous ethanol for 1 hour respectively and then immersed in xylene for 30 minutes for clarity.

[0047] 6. After clearing, the cells were immersed in paraffin for 3 hours and embedded into blocks.

[0048] 7. The wax blocks were sliced ​​separately and prepared into cell suspensions according to patent CN 202011356471.8 A method for preparing a semi-solid cell suspension and its application. The cell suspension was prepared for immunohistochemical staining experiments; the immunohistochemical staining experiment was carried out using the LYNX480 fully automatic immunohistochemical staining machine of Tuling (Hangzhou) Biotechnology Co., Ltd. The target primary antibody (I1219) and auxiliary reagents (I20032C) used in conjunction were purchased from the commercial kit of Tuling (Hangzhou) Biotechnology Co., Ltd. The experiment adopted the conventional immunohistochemical experimental process and was carried out according to the instructions of the kit.

[0049] 8. Experimental results are as follows Figure 3 shown.

[0050] The results showed that the cleaning solution formulations with different concentration components all had good cleaning effects.

[0051] Example 4 1. Resuscitate a strain of IMR-32 cells (neuroblastoma cells, Guangzhou Keluojie KC2102) and expand the culture.

[0052] 2. 40M cells in the logarithmic growth phase were collected, with a cell viability of 95.4%; 20M cells were divided into 1 group and divided into 2 groups: Group 13 and Group 14. After collecting cells from Group 13, they were resuspended in 40mL 10mM PBS and washed once.

[0053] 3. The cells in group 14 were resuspended to 40 mL using the same washing solution formula as that of group 11 in Example 3, and washed once by centrifugation at 900 RPM.

[0054] 4. The washed cells were fixed with 10% neutral formalin fixative for 2 hours, and then washed to remove the fixative.

[0055] 5. After fixation, the cells were dehydrated using 50%, 75%, 95% and anhydrous ethanol for 1 hour respectively and then immersed in xylene for 30 minutes for clarity.

[0056] 6. After clearing, the cells were immersed in paraffin for 3 hours and embedded into blocks.

[0057] 7. The wax blocks were sliced ​​separately and prepared into cell suspensions according to patent CN 202011356471.8 A method for preparing a semi-solid cell suspension and its application. The cell suspension was prepared for immunohistochemical staining experiments; the immunohistochemical staining experiments were carried out using the LYNX480 fully automatic immunohistochemical staining machine of Tuling (Hangzhou) Biotechnology Co., Ltd. The target primary antibody (I1178) and auxiliary reagents (I20032C) used in conjunction were purchased from the commercial kit of Tuling (Hangzhou) Biotechnology Co., Ltd. The experiment adopted the conventional immunohistochemical experimental process and was carried out according to the instructions of the kit.

[0058] 8. Experimental results are as follows Figure 4 shown.

[0059] The results showed that the quality control cleaning solution of this formula can achieve the same cleaning effect of removing staining impurities on different cell lines, which is significantly better than traditional PBS solution cleaning.

[0060] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A cell washing solution, characterized in that Includes PBS solution, glucose, EDTA and collagen.

2. A cell cleaning solution according to claim 1, characterized in that: The concentration of the PBS is 8-10 mM.

3. A cell cleaning solution according to claim 1, characterized in that: The mass volume percentage concentration of the glucose is 1%-7%.

4. A cell cleaning solution according to claim 1, characterized in that: The mass volume percentage concentration of the EDTA is 0.01%-0.07%.

5. A cell cleaning solution according to claim 1, characterized in that: The mass volume percentage concentration of the collagen is 0.005%-0.15%.

6. Use of the cell washing solution according to any one of claims 1 to 5 in the preparation of a washing solution for cell immunohistochemistry quality control products.

7. A method for preparing a cell quality control product, characterized in that: The following steps are included: (1) preparing the cell washing solution according to any one of claims 1 to 5; (2) Collect the target cells, resuspend them in cell washing solution, and then centrifuge and wash them 1-2 times; (3) The washed cells are fixed, dehydrated, embedded, and sliced ​​to prepare a semi-solid cell suspension to obtain a cell quality control product.

8. The method according to claim 7, characterized in that: The centrifugation condition is 800-1000RPM.

9. The cell quality control product prepared by the method according to any one of claims 7-8.

10. A cell immunohistochemistry detection kit, characterized in that: The invention comprises the cell washing solution according to any one of claims 1 to 5.

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