CN110646335A - A kind of sealing liquid and its application - Google Patents

Abstract

The invention relates to the technical field of sealing liquid, in particular to a sealing liquid and its application. The invention discloses a sealing solution, comprising: a first sealing solution and a second sealing solution; the first sealing solution is: Tween-20, donkey serum, bovine serum albumin and phosphate buffer solution; the second sealing solution is: Tween-20, donkey serum, bovine serum albumin, phosphate buffered saline, and polyethylene glycol octyl phenyl ether. The blocking solution provided by the invention effectively reduces the non-specific binding of FcR on the cell surface with the Fc segment of the fluorescein-conjugated antibody, and has good blocking effect, making the results of flow cytometry more accurate and less prone to deterioration.

Description

A kind of sealing liquid and its application

technical field

The invention relates to the technical field of sealing liquid, in particular to a sealing liquid and its application.

Background technique

Flow cytometry is an analytical technique that enables multiparameter, rapid quantitative analysis of single cells or other biological particles. It can analyze tens of thousands of cells at high speed, and can measure multiple parameters from one cell at the same time. It has the advantages of fast speed, high precision and good accuracy. It is one of the most advanced cell quantitative analysis technologies in the contemporary era. Quantitative analysis of cardiomyocytes by flow cytometry can obtain data information on the differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes, so as to understand the differentiation of cardiomyocytes and provide data support for the follow-up work of culturing cardiomyocytes .

Most of the fluorescein-conjugated antibodies used to label sample cells in flow cytometry are monoclonal antibodies, and a few may also be polyclonal antibodies. Compared with the relatively conservative Fc segment, the specificity of the antibody is expressed in the Fab segment. When labeling, the antigen binding site of the Fab segment is used to specifically bind to the antigen molecule on the cell, thereby labeling and relatively quantifying the expression of the antigen molecule by the cell. However, some cells express FcR (Fc receptor, Fc receptor) on the surface, such as macrophages, DCs, B lymphocytes, etc. FcR can non-specifically bind to the Fc segment of fluorescein-conjugated antibodies, which has a certain effect on the results. influences. In order to reduce the binding of non-specific proteins in flow cytometry, blocking solutions are usually used. Traditional blocking solutions include skim milk, BSA, etc. The choice of blocking solution has a very important influence on the results. Only by choosing a suitable blocking solution can an accurate and clear fluorescence analysis map be made.

The most commonly used blocking solutions are skimmed milk powder and BSA blocking solution. The BSA blocking solution is prepared from bovine serum albumin, which is expensive, and the sealing effect is not good, which seriously affects the experimental cost. The main active ingredients in skim milk powder are whey protein and casein. The components are more complex than BSA, and its sealing effect is better than that of BSA. However, skim milk powder is prone to mildew and deterioration. Combine more.

SUMMARY OF THE INVENTION

The invention provides a sealing liquid and its application, and solves the problems of poor sealing effect and easy deterioration of the existing sealing liquid.

The present invention provides a sealing solution, comprising: a first sealing solution and a second sealing solution;

The first blocking solution is: Tween-20, donkey serum, bovine serum albumin and phosphate buffered solution;

The second blocking solution is: Tween-20, donkey serum, bovine serum albumin, phosphate buffer solution and polyethylene glycol octyl phenyl ether.

Preferably, the dosage ratio of the Tween-20, the donkey serum, the bovine serum albumin and the phosphate buffer solution is: (10-100) μL: (500-5000) μL: (0.300-3.00 ) g: (9.490 to 94.90) mL, more preferably 10 μL: 500 μL: 0.300 g: 9.490 mL.

Preferably, the dosage ratio of the Tween-20, the donkey serum, the bovine serum albumin, the phosphate buffer solution and the polyethylene glycol octyl phenyl ether is: (10-100) μL : (500 to 5000) μL: (0.300 to 3.00) g: (9.390 to 93.90) mL: (100 to 1000) μL, more preferably 10 μL: 500 μL: 0.300 g: 9.390 mL: 100 μL.

The blocking solution can also be used in the present invention to inhibit the non-specific binding of the Fab segment on the cell surface and the Fc segment of the fluorescein-coupled antibody.

In the present invention, the cells are cardiomyocytes, 3T3, HaCat or hiPSCs, more preferably cardiomyocytes.

The present invention also provides the application of the above-mentioned blocking solution in detecting cellular proteins, comprising the following steps:

After fixing the cells, use the first blocking solution to block, and then use the second blocking solution containing the primary antibody to form the first complex;

adding the second blocking solution containing the secondary antibody to the first complex to form a second complex;

The second complex is detected.

In the present invention, the primary antibody is a cell monoclonal antibody, preferably a cardiomyocyte monoclonal antibody; the secondary antibody is a fluorescein-labeled goat anti-mouse IgG.

In the present invention, in the second blocking solution containing the primary antibody, the volume ratio of the primary antibody to the second blocking solution is 1:99; in the second blocking solution containing the secondary antibody, the The volume ratio of the secondary antibody to the second blocking solution was 1:999.

In the present invention, the cells are cardiomyocytes, 3T3, HaCat or hiPSCs, more preferably cardiomyocytes.

The present invention also provides an immunofluorescence staining kit, comprising the above-mentioned blocking solution.

The present invention also provides a flow cytometry detection kit, comprising the above-mentioned blocking solution.

The flow cytometry detection kit of the present invention further comprises: a fixative, a cardiomyocyte monoclonal antibody and a fluorescein-labeled goat anti-mouse IgG.

The present invention also provides a cardiomyocyte protein immunoblotting kit, comprising the above-mentioned blocking solution.

The reagents other than the blocking solution provided in the immunofluorescence staining kit, flow cytometry detection kit and western blotting kit provided by the present invention are all reagents well known to those skilled in the art, which are not particularly limited in the present invention.

As can be seen from the above technical solutions, the present invention has the following advantages:

The invention provides a blocking solution, comprising: a first blocking liquid and a second blocking liquid; the first blocking liquid is: Tween-20, donkey serum, bovine serum albumin and phosphate buffer solution; the second blocking liquid is: Tween-20, donkey serum, bovine serum albumin, phosphate buffered saline, and polyethylene glycol octyl phenyl ether.

The blocking solution provided by the invention effectively reduces the non-specific binding of FcR on the cell surface with the Fc segment of the fluorescein-coupled antibody, and the blocking effect is good, so that the results of flow cytometry are more accurate, and it is not easy to deteriorate, and the experimental cost is saved .

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.

Fig. 1 is the cardiomyocyte flow cytometry distribution bright field diagram that the comparative example 1 of the present invention provides;

Fig. 2 is the cardiomyocyte flow cytometry distribution fluorescence diagram provided by Comparative Example 1 of the present invention;

Fig. 3 is the cardiomyocyte flow cytometry analysis diagram provided by Comparative Example 1 of the present invention;

FIG. 4 is a bright field diagram of flow cytometry distribution of cardiomyocytes provided in Example 1 of the present invention;

Fig. 5 is the cardiomyocyte flow cytometry distribution fluorescence diagram provided in Example 1 of the present invention;

FIG. 6 is a flow cytometric analysis diagram of cardiomyocytes provided in Example 1 of the present invention.

Detailed ways

In order to make the purpose, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the embodiments described below are only a part of the implementation of the present invention. examples, but not all examples. 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.

In the embodiment of the present invention, FlowBuffer-1: take 10ml as an example: 10μl Tween-20, 500μl donkeyserum, 0.300g BSA, 9.490ml PBS;

FlowBuffer-2: Take 10ml as an example: 10μl Tween-20, 500μl donkey serum, 0.300g BSA, 100μl Triton X-100, 9.390ml PBS.

In the comparative example of the present invention, FlowBuffer-1: Take 10ml as an example: 0.100g BSA, 10.000ml PBS;

FlowBuffer-2: Take 10ml as an example: 0.300g BSA, 100μl Triton X-100, 9.900ml PBS.

In the examples of the present invention, RPMI 1640 basal medium and 2% insulin-free cell culture additive B27 are referred to as RPMI1640+B27minus insulin for short.

The reagents used in the examples of the present invention are all commercially available, wherein, hiPSC is specifically hiPSC-U1, which was purchased from Beijing Saibei Co., Ltd., CA4002106; TeSR-E8 medium was purchased from Stem Cell, cat.05990; RPMI 1640 was purchased from from Gibco, 11875; B 27minus insulin from Gibco, A18956; B 27supplement from Gibco, 17504; methanol from Tianjin Damao Chemical Reagent Factory, 2305; paraformaldehyde from meilunbio, MA0192; 0.25% (wt/vol ) trypsin-EDTA was purchased from Gibco, 25200; FBS was purchased from Gibco, A3160901; 0.22 μm filter was purchased from MILLEX, SLGP033RB; Matrigel was purchased from CORNING, 354277; Triton X-100 was purchased from SLBW6818, SIGMA; the primary antibody was cardiac muscle calcium Protein (cTnT) was purchased from Abcam; secondary antibody was Goat Anti-Mouse IgG H&L, purchased from Abcam; donkey serum was purchased from Beijing Soleibo Technology Co., Ltd., SL050.

Example 1

In this example, human induced pluripotent stem cells (hiPSCs) are induced to differentiate into cardiomyocytes

1. Cultivation method of hiPSC-U1: Put Matrigel Matrigel from -80 ℃ into 4 ℃ overnight freeze-thaw, the next day, divide Matrigel and add 100 μl of Matrigel to 10 ml of DMEM/F-12 to culture based on 15 ml centrifuge tube, store it in 15 ml centrifuge tube. Store in a 4°C refrigerator for later use. Every day, each well was replaced with a new medium: 4 ml of TeSR-E8 medium, until the number of hiPSCs reached about 75%.

2. On the 0th day of differentiation, discard the old medium, replace and add 4ml (12μM CHIR99021+RPMI/B-27minus insulin) medium to each well, put the multi-well plate back to 37°C, and incubate in a 5% _CO2 incubator for 24h .

3. On the first day of differentiation, discard the old medium, add 4 ml of RPMI/B-27 medium (without insulin) to each well, and put the multi-well plate back in a 37°C, 5% CO ₂ incubator for 24 hours.

4. The third day of differentiation, 72h after adding CHIR 99021. Taking one well as an example, use a 15ml pipette to collect 2ml of old medium from the well to prepare a combined medium Configuration method: Mix 2ml of old medium with 2ml of (RPMI/B-27minusinsulin). 4 [mu]l (5 mM IWP 2) was added to 4 ml of combined medium. Before discarding the remaining 2 ml of medium, gently rock the plate back and forth to suspend the cell debris, making sure to discard the cell debris by aspiration. 4 ml of combination medium containing IWP 2 was added to each well. Repeat the procedure to change the medium for several other wells.

5. On the 5th day of differentiation, the old medium was discarded, 4 ml (RPMI/B-27minus insulin) medium was added to each well, and the multi-well plate was placed back in a 37°C, 5% CO ₂ incubator for 24 hours.

6. On the 7th day of differentiation and every 3 days thereafter, discard the old medium, replace with 4ml of RPMI/B-27 medium per well, and put the multi-well plate back into the 37°C, 5% _CO2 incubator Incubate for 24h.

Example 2

This example is the immunofluorescence staining of cardiomyocytes in Example 1

(1) Digestion:

Aspirate the old medium and wash the differentiated cells twice with 2 ml of PBS per well. The PBS was aspirated, and 2 ml of (0.25% (wt/vol) trypsin-EDTA) dissociation enzyme was added to dissociate the cells, and the cells were digested at 37° C. in a 5% incubator for 5 min.

(2) Filter cells:

Cells were singulated by pipetting 5-10 times using a 1 ml pipette. The pipetted single cell suspension was then transferred to a 15 ml centrifuge tube containing 4 ml of RPMI 20 and the cells were naturally filtered by gravity using a 70 μm cell sieve.

(4) Count:

Take 10 μl of the cell suspension to count the cells with a hemocytometer; centrifuge the cell suspension at room temperature for 4 min at a speed of 1000 r/min. After centrifugation, the supernatant was discarded using a 1 ml pipette.

(5) Cell fixation:

After the old medium was discarded by centrifugation, 1 ml of 4% (v/v) paraformaldehyde was added to resuspend the cell pellet and incubated at room temperature for 15 min.

(6) Cell closure:

Add 1×10 ⁶ cells into a 15 ml centrifuge tube containing 2 ml of FlowBuffer-1, centrifuge at 200 g for 5 min at room temperature, and discard the supernatant. Repeat washing 2 times.

(7) Incubation of primary antibody:

According to the volume ratio of cTnT:FlowBuffer-2=1μl:99μl, resuspend the cell pellet in FlowBuffer-2 diluted 100μl of primary antibody, add it to the cell pellet, pipette evenly, and incubate the mixture at room temperature for 1h or at 4°C Incubate overnight.

(8) Incubation of primary antibody:

Wash the cells with 2ml of FlowBuffer-2, and configure them according to the volume ratio of Goat Anti-Mouse IgG H&L:FlowBuffer-2=1μl:999μl, and resuspend the cell pellet in 100μl of FlowBuffer-2 containing secondary antibody to obtain a mixture. The mixture was incubated for 30 min at room temperature in the dark.

Comparative Example 1

This comparative example is the immunofluorescence staining of cardiomyocytes in Example 1

The difference between this comparative example and Example 2 is: FlowBuffer-1: 0.020g BSA, 2.000ml PBS; FlowBuffer-2: 0.020g BSA, 20μl Triton X-100, 1.980ml PBS.

Example 3

In this example, flow cytometry was performed on the cardiomyocytes after immunofluorescence staining in Example 2 and Comparative Example 1.

Cardiomyocytes incubated in Example 2 and Comparative Example 1 were washed twice with 2 ml of FlowBuffer-2 respectively; the cell pellet was resuspended in 50 μl of FlowBuffer-1 and transferred to a flow round bottom tube. The flow tubes were placed on ice and flow cytometric analysis was performed with FACS Calibur. The results are shown in Figures 1-6.

From the comparative analysis of Figures 1-3 and Figures 4-6, it can be seen that the cells obtained by the closed liquid shot in Comparative Example 1 are relatively small, and the fluorescence detection value is relatively low, so the amount of cells required for the experiment will deviate from the experimental theoretical value; There are many cells obtained by the liquid shot, which is not much different from the expected experimental theoretical value, and is approximately close to the corresponding cell density.

As mentioned above, the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: The technical solutions described in the embodiments are modified, or some technical features thereof are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A sealant fluid, comprising: a first confining liquid and a second confining liquid;

the first confining liquid is: tween-20, donkey serum, bovine serum albumin and phosphate buffer solution;

the second confining liquid is: tween-20, donkey serum, bovine serum albumin, phosphate buffer solution and polyethylene glycol octyl phenyl ether.

2. The sealing solution according to claim 1, wherein the tween-20, the donkey serum, the bovine serum albumin and the phosphate buffer solution are used in a ratio of: (10-100) μ L: (500-5000) μ L: (0.300-3.00) g: (9.490-94.90) mL.

3. The sealing solution according to claim 1, wherein the tween-20, the donkey serum, the bovine serum albumin, the phosphate buffer solution and the polyethylene glycol octyl phenyl ether are used in a ratio of: (10-100) μ L: (500-5000) μ L: (0.300-3.00) g: (9.390-93.90) mL: (100-1000) mu L.

4. Use of a blocking solution according to any one of claims 1 to 3 for inhibiting non-specific binding of a Fab fragment on the cell surface to an Fc fragment of a fluorescein-conjugated antibody.

5. The use according to claim 4, wherein the cell is a cardiomyocyte, 3T3, HaCat or hipSC.

6. Use of a blocking solution according to any one of claims 1 to 3 for the detection of cellular proteins, comprising the steps of:

after the cells are fixed, sealing by adopting a first sealing liquid, and then adopting a second sealing liquid containing a primary antibody to form a first compound;

adding the second blocking solution containing an anti-antibody to the first complex to form a second complex;

detecting the second complex.

7. The use according to claim 6, wherein the cell is a cardiomyocyte, 3T3, HaCat or hipSC.

8. An immunofluorescent staining kit comprising the blocking solution according to any one of claims 1 to 3.

9. A flow cytometry kit comprising the blocking solution of any one of claims 1 to 3.

10. A protein immunoblotting kit comprising the blocking solution according to any one of claims 1 to 3.

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