CN107328620B - Blocking buffer solution and kit for flow cytometry - Google Patents
Blocking buffer solution and kit for flow cytometry Download PDFInfo
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- CN107328620B CN107328620B CN201710484073.6A CN201710484073A CN107328620B CN 107328620 B CN107328620 B CN 107328620B CN 201710484073 A CN201710484073 A CN 201710484073A CN 107328620 B CN107328620 B CN 107328620B
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a blocking buffer solution for flow cytometry, which comprises a human immunoglobulin solution, a mouse immunoglobulin solution, a rat immunoglobulin solution, a hamster immunoglobulin solution and a bovine serum albumin solution, wherein the human immunoglobulin solution comprises human immunoglobulin, sodium azide and a phosphate buffer solution; the mouse immunoglobulin solution comprises mouse immunoglobulin, sodium azide and phosphate buffer; the rat immune globulin solution comprises rat immune globulin, sodium azide and phosphate buffer solution; the hamster immunoglobulin solution comprises hamster immunoglobulin, sodium azide and phosphate buffer; the bovine serum albumin solution comprises bovine serum albumin, sodium azide and phosphate buffer solution; when in use, the solutions are mixed again. The invention can provide a rapid and efficient sealing effect for the flow cytometry, greatly reduce the interaction of nonspecific protein and enhance the dyeing effect.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a sealing buffer solution and a kit for flow cytometry.
Background
The flow cytometry is a technology for carrying out multi-parameter and rapid quantitative analysis and sorting on single cells by a monoclonal antibody on a cellular molecular level by utilizing a flow cytometer, has the advantages of high speed, high precision and good accuracy, and is one of modern advanced cell quantitative analysis technologies. Therefore, the method is extremely widely applied to the fields of medicine and scientific research.
With the continuous progress of science and technology, the mass spectrometry flow cytometry which inherits the characteristics of high-speed analysis of the traditional flow cytometry and has high resolution capability of mass spectrometry detection gradually becomes a new development direction in the technical field of flow cytometry, and has wide application prospect in the research of a plurality of fields such as hematopoiesis, immunity, stem cells, cancers, drug screening and the like.
However, in both the conventional flow cytometry and mass spectrometry, during the antibody-antigen reaction, due to the high antibody concentration or the specific cross reaction between the antibody and the antigen, non-specific binding is inevitably generated, resulting in a high background, which further affects the precise analysis of experimental data. This also requires more stringent and elaborate processing of the flow cytometric test samples.
In order to prevent or reduce non-specific interactions between antibody antigens, the antibody incubation process is typically started after the cells are pretreated with blocking buffer. Therefore, the blocking buffer is particularly important during the sample preparation process.
In the practical use process of the existing commercial blocking buffer, although the false positive phenomenon possibly occurring in antibody staining is reduced to a certain extent, with the further development of flow cytometry and more accurate flow data analysis, certain limitations still exist. As shown in FIG. 1 (panels A and B), the non-specific binding rate was 0.99% when no blocking buffer was added to panel A, and 0.85% when a commercially available blocking buffer (Human TruStain FcXTM, Biolegend) was added to panel B.
Disclosure of Invention
The invention aims to provide a blocking buffer solution and a kit for flow cytometry, so as to overcome the defects of the prior art.
The invention adopts the following technical scheme:
a blocking buffer solution for flow cytometry comprises human immunoglobulin solution, mouse immunoglobulin solution, rat immunoglobulin solution, hamster immunoglobulin solution, and bovine serum albumin solution,
the human immunoglobulin solution comprises human immunoglobulin, sodium azide and phosphate buffer;
the mouse immunoglobulin solution comprises mouse immunoglobulin, sodium azide and phosphate buffer;
the rat immune globulin solution comprises rat immune globulin, sodium azide and phosphate buffer solution;
the hamster immunoglobulin solution comprises hamster immunoglobulin, sodium azide and phosphate buffer;
the bovine serum albumin solution comprises bovine serum albumin, sodium azide and phosphate buffer solution;
when in use, the human immunoglobulin solution, the mouse immunoglobulin solution, the rat immunoglobulin solution, the hamster immunoglobulin solution and the bovine serum albumin solution are mixed again.
Further, the human immunoglobulin solution comprises 15-25 parts by mass of human immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution, wherein the parts by mass are measured in mg and the parts by volume are measured in ml.
Further, the mouse immunoglobulin solution comprises 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution, wherein the parts by mass are measured in mg and the parts by volume are measured in ml.
Further, the rat immunoglobulin solution comprises 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution, wherein the parts by mass are measured in mg and the parts by volume are measured in ml.
Further, the hamster immunoglobulin solution comprises 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 part by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffer solution, wherein the parts by mass are in mg units and the parts by volume are in ml units.
Further, the bovine serum albumin solution comprises 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide and 75-125 parts by volume of phosphate buffer solution, wherein the parts by mass are mg as a metering unit, and the parts by volume are ml as a metering unit.
Further, when used, the volume ratio of human immunoglobulin solution, mouse immunoglobulin solution, rat immunoglobulin solution, hamster immunoglobulin solution, and bovine serum albumin solution is 1:1:1:1: 100.
Further, the human immunoglobulin solution comprises 15-25 parts by mass of human immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the mouse immunoglobulin solution comprises 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the rat immune globulin solution comprises 15-25 parts by mass of rat immune globulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the hamster immunoglobulin solution comprises 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the bovine serum albumin solution comprises 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide and 75-125 parts by volume of phosphate buffer solution;
when in use, the volume ratio of the human immunoglobulin solution, the mouse immunoglobulin solution, the rat immunoglobulin solution, the hamster immunoglobulin solution and the bovine serum albumin solution is 1:1:1: 100;
wherein, the mass part is mg as a metering unit, and the volume part is ml as a metering unit.
Further, the human immunoglobulin solution, the mouse immunoglobulin solution, the rat immunoglobulin solution, the hamster immunoglobulin solution and the bovine serum albumin solution were mixed well and stored at 2 to 8 ℃.
A blocking kit for flow cytometry comprises any one of the blocking buffers for flow cytometry.
The invention has the beneficial effects that:
the blocking buffer solution for the flow cytometry comprises a human immunoglobulin solution, a mouse immunoglobulin solution, a rat immunoglobulin solution, a hamster immunoglobulin solution and a bovine serum albumin solution, can provide a quick and efficient blocking effect for the flow cytometry, greatly reduces the interaction of nonspecific proteins, can efficiently avoid the nonspecific binding of antibody and antigen, reduces the false positive probability and enhances the dyeing effect.
Drawings
FIG. 1 is a graph showing the results of the nonspecific binding rates of the antibodies of each group in example 1.
Detailed Description
The invention is explained in more detail below with reference to exemplary embodiments and the accompanying drawings. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
A blocking buffer solution for flow cytometry comprises human immunoglobulin solution, mouse immunoglobulin solution, rat immunoglobulin solution, hamster immunoglobulin solution, and bovine serum albumin solution,
the human immunoglobulin solution comprises 15-25 parts by mass of human immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the mouse immunoglobulin solution comprises 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the rat immune globulin solution comprises 15-25 parts by mass of rat immune globulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the hamster immunoglobulin solution comprises 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the bovine serum albumin solution comprises 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide and 75-125 parts by volume of phosphate buffer solution;
fully mixing human immunoglobulin solution, mouse immunoglobulin solution, rat immunoglobulin solution, hamster immunoglobulin solution and bovine serum albumin solution, and respectively storing at 2-8 deg.C; when in use, the human immunoglobulin solution, the mouse immunoglobulin solution, the rat immunoglobulin solution, the hamster immunoglobulin solution and the bovine serum albumin solution are mixed again, and the volume ratio of the human immunoglobulin solution to the mouse immunoglobulin solution to the bovine serum albumin solution is 1:1:1: 100;
wherein, the mass part is mg as a metering unit, and the volume part is ml as a metering unit.
A blocking kit for flow cytometry comprises the blocking buffer for flow cytometry.
The following examples relate to human immunoglobulin powder (Equisech-Bio), mouse immunoglobulin powder (Equisech-Bio), rat immunoglobulin powder (Equisech-Bio), hamster immunoglobulin powder (Equisech-Bio), bovine serum albumin powder (Equisech-Bio), sodium azide powder (Sigma), phosphate buffer (pH7.4, Gibco), FITC-CD3e antibody (OKT3, Biolegend), APC-CD19 antibody (HIB19, Biolegend).
Example 1
1) Human immunoglobulin solution: weighing 20mg human immunoglobulin powder into a clean centrifuge tube, weighing 0.2mg sodium azide powder into the centrifuge tube, adding 1ml phosphate buffer solution, mixing well, and storing at 2-8 ℃.
2) Mouse immunoglobulin solution: weighing 20mg of mouse immunoglobulin powder into a clean centrifugal tube, weighing 0.2mg of sodium azide powder into the centrifugal tube, adding 1ml of phosphate buffer solution, fully mixing, and storing at 2-8 ℃.
3) Rat immunoglobulin solution: weighing 20mg rat immunoglobulin powder into a clean centrifuge tube, weighing 0.2mg sodium azide powder into the centrifuge tube, adding 1ml phosphate buffer solution, mixing well, and storing at 2-8 ℃.
4) Hamster immunoglobulin solution: weighing 20mg hamster immunoglobulin powder into a clean centrifuge tube, weighing 0.2mg sodium azide powder into the centrifuge tube, adding 1ml phosphate buffer solution, mixing well, and storing at 2-8 deg.C.
5) Bovine serum albumin solution: weighing 500mg bovine serum albumin powder into a clean centrifuge tube, weighing 20mg sodium azide powder into the centrifuge tube, adding 100ml phosphate buffer solution, mixing well, and storing at 2-8 ℃.
6) 300ul of bovine serum albumin solution was taken to resuspend 3x10^6 human Peripheral Blood Mononuclear Cells (PBMC) and divided equally into three groups A, B and C, each group being 100 ul.
7) Group A was used as a blank control without addition of blocking buffer; group B5 ul of the existing commercial blocking buffer (HumantUStain FcXTM, Biolegend) was added; and adding 1ul of human immunoglobulin solution, 1ul of mouse immunoglobulin solution, 1ul of rat immunoglobulin solution and 1ul of hamster immunoglobulin solution into the group C, respectively mixing uniformly, and incubating on ice for 20 min.
8) 1ul FITC-CD3e antibody and 1ul APC-CD19 antibody are added into each component respectively, and after being fully mixed, the mixture is placed on ice for incubation for 30 min.
9) Cells were washed by adding 1ml bovine serum albumin solution to each group, 350g/5min, and the supernatant was discarded.
10) 1ml of bovine serum albumin solution was added to each group to wash the cells at 350g/5min, and the supernatant was discarded.
11) Each group was resuspended in 200ul of bovine serum albumin solution, the flow cytometer cytoflex (Beckman Coulter) was turned on, and the three groups were statistically analyzed.
The results are shown in FIG. 1. The CD3e + and CD19+ double positive cell populations are not present in nature. As can be seen from fig. 1, in group a, the cell population double positive for CD3e + and CD19+ was 0.99% without addition of blocking buffer, and the non-specific binding rate was high. In group B, the CD3e + and CD19+ double positive cell population accounted for 0.85% with the addition of the current commercial blocking buffer. Group C under the blocking conditions of the present invention, the double positive cell population of CD3e + and CD19+ accounted for 0.24%. The non-specific binding rate is obviously reduced after the blocking buffer solution is adopted, and the blocking effect is far better than that of the existing commercial blocking buffer solution.
Claims (3)
1. A blocking buffer solution for flow cytometry is characterized by comprising a human immunoglobulin solution, a mouse immunoglobulin solution, a rat immunoglobulin solution, a hamster immunoglobulin solution and a bovine serum albumin solution,
the human immunoglobulin solution comprises 15-25 parts by mass of human immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the mouse immunoglobulin solution comprises 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the rat immune globulin solution comprises 15-25 parts by mass of rat immune globulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the hamster immunoglobulin solution comprises 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 part by mass of sodium azide and 0.75-1.25 parts by volume of phosphate buffer solution;
the bovine serum albumin solution comprises 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide and 75-125 parts by volume of phosphate buffer solution;
when in use, the human immunoglobulin solution, the mouse immunoglobulin solution, the rat immunoglobulin solution, the hamster immunoglobulin solution and the bovine serum albumin solution are mixed again according to the volume ratio of 1:1:1:1: 100;
wherein, the mass part is mg as a metering unit, and the volume part is ml as a metering unit.
2. The blocking buffer according to claim 1, wherein the human immunoglobulin solution, the mouse immunoglobulin solution, the rat immunoglobulin solution, the hamster immunoglobulin solution, and the bovine serum albumin solution are mixed well and stored at 2-8 ℃.
3. A blocking kit for flow cytometry comprising the blocking buffer for flow cytometry according to any one of claims 1 to 2.
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Denomination of invention: Blocked buffer and reagent kit for flow cytometry Effective date of registration: 20230519 Granted publication date: 20200605 Pledgee: Industrial and Commercial Bank of China Limited Hangzhou Yuhang sub branch Pledgor: ZHEJIANG PLTTECH HEALTH TECHNOLOGY CO.,LTD. Registration number: Y2023330000939 |
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