CN113218847A - Hemolytic agent for flow cytometry analysis and preparation method and application method thereof - Google Patents
Hemolytic agent for flow cytometry analysis and preparation method and application method thereof Download PDFInfo
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- 239000003219 hemolytic agent Substances 0.000 title claims abstract description 106
- 238000000684 flow cytometry Methods 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims description 19
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 83
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 68
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 claims abstract description 62
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims abstract description 60
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 49
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 34
- 229950000244 sulfanilic acid Drugs 0.000 claims abstract description 29
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 27
- 235000019270 ammonium chloride Nutrition 0.000 claims abstract description 26
- 239000012091 fetal bovine serum Substances 0.000 claims abstract description 24
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 22
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims abstract description 21
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 16
- 239000007853 buffer solution Substances 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims description 24
- 239000008363 phosphate buffer Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 11
- 239000000706 filtrate Substances 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 11
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 11
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 11
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 11
- 239000012894 fetal calf serum Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 230000006037 cell lysis Effects 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 7
- 210000004369 blood Anatomy 0.000 claims description 7
- 239000008280 blood Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000005336 cracking Methods 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 229960001484 edetic acid Drugs 0.000 claims description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 29
- 210000004027 cell Anatomy 0.000 abstract description 28
- 230000009089 cytolysis Effects 0.000 abstract description 28
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 abstract description 20
- 230000001205 effect on erythrocytes Effects 0.000 abstract description 7
- 230000000052 comparative effect Effects 0.000 description 19
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 12
- 238000012795 verification Methods 0.000 description 10
- 239000011734 sodium Substances 0.000 description 7
- 210000004698 lymphocyte Anatomy 0.000 description 6
- 239000006166 lysate Substances 0.000 description 6
- 210000005259 peripheral blood Anatomy 0.000 description 6
- 239000011886 peripheral blood Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 210000001616 monocyte Anatomy 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 210000003714 granulocyte Anatomy 0.000 description 4
- CBOIHMRHGLHBPB-UHFFFAOYSA-N hydroxymethyl Chemical compound O[CH2] CBOIHMRHGLHBPB-UHFFFAOYSA-N 0.000 description 4
- 210000000265 leukocyte Anatomy 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 206010018910 Haemolysis Diseases 0.000 description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- 229960002897 heparin Drugs 0.000 description 3
- 229920000669 heparin Polymers 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000441 effect on granulocytes Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000003228 hemolysin Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
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- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a hemolytic agent for flow cytometry analysis. It comprises phosphate buffer solution (PBS buffer solution), ammonium chloride (NH)4Cl), disodium ethylenediaminetetraacetate (EDTA-Na)2) Bovine Serum Albumin (BSA), sulfanilic acid, fetal bovine serum, and formaldehyde (CH)2O) and ethylene glycol ((CH)2OH)2). The invention has the advantages of good lysis effect on erythrocytes, complete erythrocyte lysis, good cell grouping effect, good stability of the sample after lysis and high detection efficiency. The invention also discloses a method for preparing the hemolytic agent for flow cytometry. The invention also discloses an application method of the hemolytic agent for flow cytometry.
Description
Technical Field
The present invention relates to a hemolytic agent for flow cytometry analysis. The invention also relates to a method for preparing the hemolytic agent for flow cytometry. The invention also relates to an application method of the hemolytic agent for flow cytometry.
Background
The existing hemolytic agent formula is generally a universal formula and has the following disadvantages: the lysis effect of the red blood cells is poor, the incomplete lysis phenomenon of the red blood cells often occurs, and the grouping effect of the cells is poor; the stability of the sample after cracking is poor, or the target components in the blood are damaged, so that the detection result is deviated.
Therefore, it is necessary to develop a hemolytic agent which has a good lysis effect on erythrocytes, a good cell grouping effect, and a good stability of a sample after lysis.
Disclosure of Invention
The first purpose of the invention is to provide a hemolytic agent for flow cytometry analysis, which has a good lysis effect on erythrocytes, can completely lyse the erythrocytes, has a good cell grouping effect, and has good stability and high detection efficiency of a lysed sample.
The second object of the present invention is to provide a method for preparing the hemolytic agent for flow cytometry.
The third purpose of the invention is to provide an application method of the hemolytic agent for flow cytometry.
In order to achieve the first object of the present invention, the technical solution of the present invention is: a hemolytic agent for flow cytometry, characterized in that: comprises phosphate buffer solution (PBS buffer solution), ammonium chloride (NH)4Cl), disodium ethylenediaminetetraacetate (EDTA-Na)2) Bovine Serum Albumin (BSA), sulfanilic acid, fetal bovine serum, and formaldehyde (CH)2O) and ethylene glycol ((CH)2OH)2)；
Wherein the dosage of each component is as follows,
in the technical scheme, the dosage of each component is,
in the technical scheme, the dosage of each component is,
in order to achieve the second object of the present invention, the technical solution of the present invention is: the method for preparing the hemolytic agent for flow cytometry is characterized by comprising the following steps: comprises the following steps of (a) carrying out,
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: adding ammonium chloride (NH)4Cl), disodium ethylenediaminetetraacetate (EDTA-Na)2) Bovine Serum Albumin (BSA), sulfanilic acid;
step three: adding fetal calf serum and formaldehyde (CH)2O), ethylene glycol ((CH)2OH)2)；
Step four: filtering, and storing to obtain filtrate as hemolytic agent.
In the technical scheme, in the second step, 8.1g of ammonium chloride, 0.043g of ethylene diamine tetraacetic acid, 15g of bovine serum albumin and 5.5g of sulfanilic acid are added;
in step three, 6mL of fetal bovine serum and formaldehyde (CH) are added2O)12mL, ethylene glycol ((CH)2OH)2)25mL。
In order to achieve the three objects of the invention, the technical scheme of the invention is as follows: the application method of the hemolytic agent for flow cytometry is characterized in that: applying the hemolytic agent for flow cytometry to red blood cell lysis in human blood;
the hemolytic agent for flow cytometry is applied to red blood cell lysis in human blood and comprises the following steps,
the method comprises the following steps: preparation of reagents:
diluting 10X hemolytic agent into 1X hemolytic agent, and storing at room temperature for later use;
step two: a cracking step:
s21: 1 × hemolytic agent is placed at room temperature for a period of time before use until the temperature of the solution rises to room temperature;
s22: adding 2mL of 1 Xhemolytic agent into each 100 mu L of detection sample, mixing uniformly, and immediately vortex and shake for 2 seconds;
s23: incubating the mixed solution prepared in the S22 at room temperature in a dark place;
s24: centrifuging the mixed solution incubated in the S23 at room temperature;
s25: removing the supernatant centrifuged in S24 by suction, and adding 1 × hemolytic agent into the flow tube;
s26: standing the mixed solution prepared in the step S25 at room temperature in a dark place;
s27: centrifuging the mixed solution prepared in the step S26 at room temperature;
s28: removing the supernatant after centrifugation in S27, adding PBS buffer solution (phosphate buffer solution) into the flow tube, and mixing uniformly to obtain an analysis preparation;
s29: analyzing the detection sample by an up-flow cytometer.
In the above-described embodiment, the analytical preparation prepared in step S28 is stored in a dark place at 2 to 8 ℃ for flow cytometry analysis within 24 hours before flow cytometry analysis.
In the technical scheme, in S23, incubating the mixed solution at room temperature for 20 minutes in a dark place;
in S26, the mixed solution was left standing at room temperature for 10min in the dark.
In the technical scheme, in S24, the incubated mixed solution is centrifuged for 5min at room temperature, and the centrifugation speed is 1500 rpm;
in S27, the mixed solution was centrifuged at 1500rpm for 5min at room temperature.
In the technical scheme, 0.5mL of 1 Xhemolytic agent is added into a flow tube in S25;
in S28, 0.5ml pbs buffer (phosphate buffer) was added to the flow tube.
The invention has the following advantages:
(1) the invention contains protein protective agent BSA and fetal calf serum, contains glycol for keeping the cell morphology from being damaged, and has the effect of enhancing the stability of sulfanilic acid;
(2) the invention has good lysis effect on erythrocytes, complete erythrocyte lysis, good cell grouping effect, good stability of the sample after lysis and high detection efficiency.
Drawings
FIG. 1 is a graph showing the results of the experimental group of the experimental verification 1 of the present invention.
FIG. 2 is a graph showing the results of the control group of the test of the present invention, verification 1.
FIG. 3 is a graph showing the results of the experimental group of the experimental verification 2 of the present invention.
FIG. 4 is a graph showing the results of the control group of the test of the present invention, verification 2.
Fig. 5 is a graph showing the results of the experimental group of the experimental verification 3 of the present invention.
FIG. 6 is a graph showing the results of the control group of the test of the present invention, verification 3.
FIG. 7 is a flow chart of a process for preparing a hemolytic agent for flow cytometry according to the present invention.
In both fig. 1 and fig. 2, P1 is the leukocyte proportion; p2 is the ratio of erythrocytes.
Detailed Description
The embodiments of the present invention will be described in detail with reference to the accompanying drawings, which are not intended to limit the present invention, but are merely exemplary. While the advantages of the invention will be clear and readily understood by the description.
A hemolytic agent for flow cytometry analysis comprises PBS buffer solution and NH4Cl、EDTA-Na2BSA, sulfanilic acid, fetal bovine serum, CH2O and (CH)2OH)2；
Wherein the dosage of each component is as follows,
furthermore, the amount of each component is,
furthermore, the amount of each component is,
the method for preparing the hemolytic agent for flow cytometry comprises the following steps,
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: 8.1g of NH were added4Cl、0.043g EDTA-Na215g BSA, 5.5g sulfanilic acid;
step three: 6mL fetal bovine serum, 12mL CH were added2O、25mL(CH2OH)2；
Step four: filtering, and storing to obtain filtrate as hemolytic agent.
The application method of the hemolytic agent for flow cytometry is characterized in that the hemolytic agent for flow cytometry is applied to erythrocyte lysis in human blood;
the hemolytic agent for flow cytometry is applied to red blood cell lysis in human blood and comprises the following steps,
the method comprises the following steps: preparation of reagents:
diluting the 10X hemolytic agent to a 1X hemolytic agent, for example: adding 5mL of 10X hemolytic agent for flow cytometry analysis into 45mL of deionized water, mixing uniformly to prepare 1X hemolytic agent, storing at room temperature for later use, and storing 1X hemolytic agent at 2-8 deg.C for 1 month;
step two: a cracking step:
s21: 1x hemolytic agent is required to be placed for a period of time at room temperature before use until the temperature of the solution rises to room temperature;
s22: adding 2mL of 1 × hemolytic agent into each 100 μ L of detection sample (labeled or other methods, and the prior art), mixing, and immediately vortexing for 2 seconds;
s23: incubating the mixed solution prepared in the S22 at room temperature in a dark place;
s24: centrifuging the mixed solution incubated in the S23 at room temperature;
s25: removing the supernatant centrifuged in S24 by suction, and adding 1 × hemolytic agent into the flow tube;
s26: standing the mixed solution prepared in the step S25 at room temperature in a dark place;
s27: centrifuging the mixed solution prepared in the step S26 at room temperature;
s28: removing the supernatant after centrifugation in S27, adding PBS buffer solution (phosphate buffer solution) into the flow tube, and mixing uniformly to obtain an analysis preparation;
s29: analyzing the detection sample by an up-flow cytometer.
Further, in any case, the analytical preparation prepared in step S28 is stored at 2 to 8 ℃ in the dark before flow cytometry analysis, and flow cytometry analysis should be performed within 24 hours.
Further, in S23, the mixed solution was incubated at room temperature for 20 minutes in the absence of light;
in S26, the mixed solution was left standing at room temperature for 10min in the dark.
Further, in S24, the incubated mixed solution was centrifuged at 1500rpm for 5min at room temperature;
in S27, the mixed solution was centrifuged at 1500rpm for 5min at room temperature.
Further, in S25, 0.5mL of 1 × hemolytic agent was added to the flow tube;
in S28, 0.5ml pbs buffer (phosphate buffer) was added to the flow tube.
Examples
Example 1
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the preparation method of the hemolytic agent for flow cytometry comprises the following steps:
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: adding ammonium chloride, disodium ethylene diamine tetraacetate, bovine serum albumin and sulfanilic acid (the components are added in sequence);
step three: adding fetal calf serum, formaldehyde and ethylene glycol (all the components are added in sequence);
step four: filtering, and storing to obtain filtrate as hemolytic agent.
Example 2
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the hemolytic agent for flow cytometry described in this example was prepared according to the same method as in example 1.
Example 3
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the hemolytic agent for flow cytometry described in this example was prepared according to the same method as in example 1.
Example 4
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the hemolytic agent for flow cytometry described in this example was prepared according to the same method as in example 1.
Example 5
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the hemolytic agent for flow cytometry described in this example was prepared according to the same method as in example 1.
Comparative example
Comparative example 3 was used as a comparative prototype with the same content, and three types (BSA, fetal bovine serum, sulfanilic acid) were selected as comparative example 1, two components (BSA, fetal bovine serum, sulfanilic acid) were contained as comparative examples 2, 3, and 4, and one component (BSA, fetal bovine serum, sulfanilic acid) was contained as comparative examples 5, 6, and 7. The same effect was obtained by selecting comparative examples using examples 1, 2, 4, 5 as comparative prototypes.
Comparative example 1
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the preparation method of the hemolytic agent for flow cytometry comprises the following steps:
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: addition of NH4Cl、EDTA-Na2(the components are added in sequence);
step three: adding formaldehyde and ethylene glycol;
step four: filtering, and storing to obtain filtrate as hemolytic agent.
And (4) conclusion: the cell lysis is incomplete, the cells are scattered, and the lysis effect is reduced after the cells are placed for a period of time.
Comparative example 2
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the preparation method of the hemolytic agent for flow cytometry comprises the following steps:
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: addition of NH4Cl、EDTA-Na2Sulfanilic acid (the components are added in sequence);
step three: adding fetal calf serum and CH2O、(CH2OH)2(the components are added in sequence);
step four: filtering, and storing to obtain filtrate as hemolytic agent.
And (4) conclusion: the lysis solution can basically lyse cells, the cells are more concentrated in clusters, but the ratio of the lysis solution to the target cells is slightly lower, and a part of the cells can be damaged.
Comparative example 3
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the preparation method of the hemolytic agent for flow cytometry comprises the following steps:
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: addition of NH4Cl、EDTA-Na2BSA, sulfanilic acid (each component is added without separation)Then);
step three: adding CH2O、(CH2OH)2(the components are added in sequence);
step four: filtering, and storing to obtain filtrate as hemolytic agent.
And (4) conclusion: the lysis solution can basically achieve the effect of cell lysis, and each component cell subset is more concentrated, but slightly less than the target cell, and part of cells can be damaged.
Comparative example 4
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the preparation method of the hemolytic agent for flow cytometry comprises the following steps:
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: addition of NH4Cl、EDTA-Na2BSA (adding all components sequentially);
step three: adding fetal calf serum and CH2O、(CH2OH)2(the components are added in sequence);
step four: filtering, and storing to obtain filtrate as hemolytic agent.
And (4) conclusion: the lysis solution can basically achieve the effect of cell lysis, each component cell subset is more concentrated, but the proportion of the cell subset to the target cells is slightly lower, partial cells can be damaged, and the lysis effect is reduced after the lysis solution is placed for a period of time.
Comparative example 5
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the preparation method of the hemolytic agent for flow cytometry comprises the following steps:
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: adding ammonium chloride and disodium ethylene diamine tetraacetate (the components are added in sequence);
step three: adding fetal calf serum, formaldehyde and ethylene glycol (all the components are added in sequence);
step four: filtering, and storing to obtain filtrate as hemolytic agent.
And (4) conclusion: the lysate can achieve a certain lysis effect on the erythrocytes, but the proportion of target cell subsets is slightly lower, a part of cells can be damaged, and the lysis effect is reduced after the lysate is placed for a period of time.
Comparative example 6
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the preparation method of the hemolytic agent for flow cytometry comprises the following steps:
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: adding ammonium chloride, disodium ethylene diamine tetraacetate, bovine serum albumin and sulfanilic acid (the components are added in sequence);
step three: adding formaldehyde and ethylene glycol (the components are added in sequence);
step four: filtering, and storing to obtain filtrate as hemolytic agent.
And (4) conclusion: the lysate can achieve a certain lysis effect on the erythrocytes, but the proportion of target cell subsets is slightly lower, a part of cells can be damaged, and the lysis effect is reduced after the lysate is placed for a period of time.
Comparative example 7
The hemolytic agent for flow cytometry analysis in this embodiment includes phosphate buffer, ammonium chloride, disodium edta, bovine serum albumin, sulfanilic acid, fetal bovine serum, formaldehyde, and ethylene glycol;
wherein the dosage of each component is as follows,
the preparation method of the hemolytic agent for flow cytometry comprises the following steps:
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: adding ammonium chloride, disodium ethylene diamine tetraacetate and sulfanilic acid (the components are added in sequence);
step three: adding formaldehyde and ethylene glycol (the components are added in sequence);
step four: filtering, and storing to obtain filtrate as hemolytic agent.
And (4) conclusion: the lysate can achieve a certain lysis effect on erythrocytes, the lysate has good stability, but the ratio of target cell subsets is obviously reduced, and the leukocyte surface protein is possibly damaged.
Through detection, the red blood cell lysis effect is good, the red blood cells are completely lysed, the cell grouping effect is good, the stability of the lysed sample is good, and the detection efficiency is high in the embodiment 3 of the invention. As can be seen from example 3 and comparative examples 1 to 7: the BSA, the fetal calf serum and the sulfanilic acid in the invention have a synergistic effect, and the BSA, the fetal calf serum and the sulfanilic acid are used together with other components and proportions in the invention according to the proportion in the invention, so that a better erythrocyte cracking effect and a better cell grouping effect can be achieved, and a cracked sample has better stability.
Test verification
Test verification 1: tests on the lytic effect of hemolytic agents on erythrocytes;
the experimental hemolytic agent (example 3) and the comparative hemolytic agent (commercially available products on the market) were used as experimental subjects, heparin anticoagulated human peripheral blood was used as experimental samples, 5 μ l of CD45-FITC (commercially available products on the market) was added to each of the experimental samples, and then the human peripheral blood was hemolyzed with each of the two hemolytic agents, and then each of the hemolytic agents was subjected to the machine test.
The results are as follows: after the hemolytic agent of the experimental group is used, the proportion of white blood cells is 89.69%, and the proportion of red blood cells is 5.95% (as shown in figure 1); after the hemolytic agent of the control group is used, the proportion of white blood cells is 64.31%, and the proportion of red blood cells is 23.88% (as shown in figure 2); the above comparison can result in: the hemolytic agent in the experimental group has better effect on the lysis of the erythrocytes.
Description of the drawings: fig. 1 and 2 are both scattergrams. In FIGS. 1 and 2, A represents the expression form selected when the particles pass through the cytometer as an area. The abscissa in fig. 1 and 2 represents the fluorescence intensity; the ordinate represents the scattered light intensity.
Test verification 2: tests on the differentiating effect of hemolytic agents on granulocytes, monocytes, lymphocytes;
the experimental hemolytic agent (example 3) and the comparative hemolytic agent (commercially available products on the market) were used as experimental subjects, heparin anticoagulated human peripheral blood was used as experimental samples, 5 μ l of CD45-FITC (commercially available products on the market) was added to each of the experimental samples, and then the human peripheral blood was hemolyzed with each of the two hemolysins, and then the results were tested on a computer.
The results are as follows: after hemolysis of the experimental group and the control group, the granulocytes, monocytes and lymphocytes of the two groups of experiments can form cell groups, but the granulocytes, monocytes and lymphocytes of the experimental group have larger distribution distance and are more obviously distinguished (as shown in figure 3); and the debris and red blood cell population of the control experiment were relatively close to the lymphocytes (as shown in figure 4). The above comparison can result in: the test group has better effect of distinguishing granulocytes, monocytes and lymphocytes after hemolysis.
Description of the drawings: fig. 3 and 4 are both scatter diagrams. In FIGS. 3 and 4, A represents the expression form selected when the particles pass through the cytometer as an area. The abscissa in fig. 3 and 4 represents the fluorescence intensity; the ordinate represents the scattered light intensity.
Test verification 3: assays for the effect of a hemolytic agent on the detection effect of a target protein;
the experimental hemolytic agent (example 3) and the comparative hemolytic agent (commercially available products in the market) were used as experimental subjects, heparin anticoagulated human peripheral blood was used as experimental samples, 5 μ l of CD3-PE-CY5.5 (commercially available products in the market) was added to each of the experimental samples, and the human peripheral blood was hemolyzed with the two hemolytic agents according to the above experimental procedures, and then separately tested by a computer.
The results are as follows: taking CD3 antigen as an example, after hemolysis, it is obvious through flow charts that two experiments can be well grouped, but the positive rate of the experimental group CD3-PE-CY5.5 is 66.46% (as shown in FIG. 5), and the positive rate of the control group CD3-PE-CY5.5 is 64.63% (as shown in FIG. 6), which indicates that the hemolytic agent in the experimental group has little influence on the detection effect of the target protein.
The above tests show that: the hemolytic agent for flow cytometry provided by the invention has a better lysis effect on erythrocytes, a better differentiation effect on granulocytes, monocytes and lymphocytes, and a smaller influence on the detection effect of target proteins.
Through detection, the same effect can be achieved by adopting the same method in other embodiments of the invention, wherein the embodiment 3 has better lysis effect on erythrocytes, better cell grouping effect and better stability of the sample after lysis.
Description of the drawings: both fig. 5 and 6 are histograms. CD3PC5.5 in FIG. 5 is an abbreviation for CD3-PE-CY 5.5. H in FIGS. 5 and 6 represents the height as the selected expression when the particles pass through the cytometer. The abscissa in fig. 5 and 6 represents the fluorescence intensity, and the ordinate represents the number.
Other parts not described belong to the prior art.
Claims (10)
1. A hemolytic agent for flow cytometry, characterized in that: comprises phosphate buffer solution, ammonium chloride, disodium ethylene diamine tetraacetate, bovine serum albumin, sulfanilic acid, fetal calf serum, formaldehyde and ethylene glycol;
wherein the dosage of each component is as follows,
。
2. A hemolytic agent for flow cytometry analysis according to claim 1, characterized in that: the dosage of each component is as follows,
。
3. A hemolytic agent for flow cytometry analysis according to claim 2, characterized in that: the dosage of each component is as follows,
。
4. A method for preparing a hemolytic agent for flow cytometry according to any one of claims 1 to 3, characterized in that: comprises the following steps of (a) carrying out,
the method comprises the following steps: preparing a phosphate buffer solution;
adding water into 1.68g of sodium dihydrogen phosphate and 5.112g of disodium hydrogen phosphate to fix the volume to 1L to obtain a phosphate buffer solution;
step two: adding ammonium chloride, disodium ethylene diamine tetraacetate, bovine serum albumin and sulfanilic acid;
step three: adding fetal calf serum, formaldehyde and ethylene glycol;
step four: filtering, and storing to obtain filtrate as hemolytic agent.
5. The method for preparing a hemolytic agent for flow cytometry according to claim 4, characterized in that: in the second step, 8.1g of ammonium chloride, 0.043g of ethylene diamine tetraacetic acid, 15g of bovine serum albumin and 5.5g of sulfanilic acid are added;
in step three, 6mL of fetal bovine serum, 12mL of formaldehyde and 25mL of ethylene glycol are added.
6. A method for using a hemolytic agent for flow cytometry, comprising: applying a hemolytic agent for flow cytometry according to any one of claims 1 to 3 to red blood cell lysis in human blood;
the hemolytic agent for flow cytometry is applied to red blood cell lysis in human blood and comprises the following steps,
the method comprises the following steps: preparation of reagents:
diluting 10X hemolytic agent into 1X hemolytic agent, and storing at room temperature for later use;
step two: a cracking step:
s21: 1 × hemolytic agent is placed at room temperature for a period of time before use until the temperature of the solution rises to room temperature;
s22: adding 2mL of 1 Xhemolytic agent into each 100 mu L of detection sample, mixing uniformly, and immediately vortex and shake for 2 seconds;
s23: incubating the mixed solution prepared in the S22 at room temperature in a dark place;
s24: centrifuging the mixed solution incubated in the S23 at room temperature;
s25: removing the supernatant centrifuged in S24 by suction, and adding 1 × hemolytic agent into the flow tube;
s26: standing the mixed solution prepared in the step S25 at room temperature in a dark place;
s27: centrifuging the mixed solution prepared in the step S26 at room temperature;
s28: removing the supernatant after centrifugation in S27, adding PBS buffer solution into the flow tube, and mixing uniformly to obtain an analysis preparation;
s29: analyzing the detection sample by an up-flow cytometer.
7. The method for applying a hemolytic agent for flow cytometry according to claim 6, wherein: before the flow cytometry analysis, the analytical preparation prepared in step S28 was stored at 2 to 8 ℃ in the dark and subjected to flow cytometry analysis over 24 hours.
8. The method for applying a hemolytic agent for flow cytometry according to claim 7, wherein: incubating the mixed solution at room temperature for 20 minutes in S23 in the absence of light;
in S26, the mixed solution was left standing at room temperature for 10min in the dark.
9. The method for applying a hemolytic agent for flow cytometry according to claim 8, wherein: in S24, centrifuging the incubated mixed solution at room temperature for 5min at the speed of 1500 rpm;
in S27, the mixed solution was centrifuged at 1500rpm for 5min at room temperature.
10. The method for applying a hemolytic agent for flow cytometry according to claim 9, characterized in that: in S25, 0.5mL of 1 × hemolytic agent was added to the flow tube;
in S28, 0.5ml pbs buffer was added to the flow tube.
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