CN113980814B - Composition for rapidly lysing peripheral red blood cells and application thereof - Google Patents
Composition for rapidly lysing peripheral red blood cells and application thereof Download PDFInfo
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- 210000003743 erythrocyte Anatomy 0.000 title claims abstract description 69
- 239000000203 mixture Substances 0.000 title claims abstract description 46
- 230000002934 lysing effect Effects 0.000 title claims abstract description 12
- 230000002093 peripheral effect Effects 0.000 title claims abstract description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 45
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 30
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 14
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 description 1
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- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- OVBJJZOQPCKUOR-UHFFFAOYSA-L EDTA disodium salt dihydrate Chemical compound O.O.[Na+].[Na+].[O-]C(=O)C[NH+](CC([O-])=O)CC[NH+](CC([O-])=O)CC([O-])=O OVBJJZOQPCKUOR-UHFFFAOYSA-L 0.000 description 1
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- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
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- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 1
- 101710160107 Outer membrane protein A Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- YVPYQUNUQOZFHG-UHFFFAOYSA-N amidotrizoic acid Chemical compound CC(=O)NC1=C(I)C(NC(C)=O)=C(I)C(C(O)=O)=C1I YVPYQUNUQOZFHG-UHFFFAOYSA-N 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 239000002280 amphoteric surfactant Substances 0.000 description 1
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- 210000003651 basophil Anatomy 0.000 description 1
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- 210000003979 eosinophil Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
Abstract
The invention provides a composition for rapidly lysing peripheral red blood cells and application thereof, wherein the composition comprises one or more of the following components: glucose, calcium chloride, sodium chloride, potassium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, DIPSO, betaine, meglumine diatrizoate, glycerol, and tween 20; the pH value is 7.0-7.6. According to the invention, the hypotonic salt solution is adopted to rapidly deplete erythrocytes, so that erythrocytes are furthest lysed in the shortest time and the non-erythrocyte activity state is maintained, and in addition, erythrocyte fragments can be far away from cell clusters during centrifugation and enrichment of leukocytes, so that the purpose of removing the cell fragments is achieved; solves the problems of complicated and long time consumption, insufficient cracking and non-erythrocyte activity reduction of the prior red cracking reagent.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a composition for quickly cracking peripheral red blood cells and application thereof.
Background
In the human body, blood is composed of various types of blood cells and other substances, mainly including erythrocytes, leukocytes, platelets, proteins, glucose, inorganic salt ions, hormones, etc., wherein the leukocytes mainly consist of lymphocytes, neutrophils, eosinophils, basophils, monocytes, etc. Modern medical diagnosis and analysis and basic science are very extensive for blood research, mainly comprising pathology prediction and diagnosis, peripheral blood stem cell culture and transplantation, blood cell sorting based on flow cytometry, cell gene expression research based on RT-PCR, detection of blood microorganisms and viruses, and the like, and furthermore, research on white blood cells to feed back physiological and pathological conditions is a common medical method, which mainly comprises immunological related inflammation and immune state assessment, leukemia research, cancer risk assessment and prediction based on circulating tumor cells, and the like. It has become extremely important for the processing of blood samples, especially for medical applications and basic research related to peripheral blood leukocyte sorting and cell culture and transplantation.
Typical blood sample pretreatment includes PBMC-based cell separation techniques, equipment-dependent blood cell separation and erythrocyte lysis techniques, with erythrocyte lysis techniques being used more widely. The PBMC separation technology has the advantages that the dosage of the used reagent is large, the price is high, the common Ficoll-Hypaque Solution separation liquid is low in cell recovery rate and complicated in operation steps in domestic comparison by import, meanwhile, the PBMC cell separation technology needs long time, the centrifuge needs to be provided with a low speed-down gear, and the requirement on equipment is high. The blood cell separation technology by means of instruments and equipment needs special separation equipment, the instrument cost is high, the standards of equipment produced by each company are inconsistent, unified operation is difficult to form, in addition, multi-sample separation is not suitable to be carried out at the same time, and cross contamination is easy. The erythrocyte lysis technology is mainly erythrocyte lysate which is developed by Saas et al in 1979 and takes ammonium chloride aqueous solution as a main component, the rest of the components are potassium bicarbonate, disodium ethylenediamine tetraacetate and the like, and the purpose of enriching white blood cells is achieved by breaking and depleting the erythrocytes. The results of cell detection by the PBMC method based on density gradient centrifugation and the erythrocyte lysis method using ammonium salt as a main component are reported in the literature to have no significant difference, but the erythrocyte lysis method is widely used due to the simplicity of operation and time saving.
The current method for cracking and depleting erythrocytes mainly depends on pH value change, salt cracking, osmotic pressure change, detergent and erythrocyte enzyme digestion reaction. The erythrocyte lysis method based on pH change and low osmotic pressure is a severe reaction, and the generated result is often variable without fixed standard, so that the end point of the erythrocyte lysis reaction is difficult to determine and control, and the damage to white blood cells is large. The salt cracking, the detergent and the enzyme digestion reaction are mild red blood cell cracking methods, and have the defects of complicated steps and long operation period, so that white blood cells are exposed in a non-cell culture environment for a long time, the damage to cells is large, and in addition, all red blood cells, especially nucleated red blood cells, are difficult to remove.
The 201880039682.3 patent uses high concentration salt solution and fixative (formaldehyde-containing solution) to increase the solution tension, so that the cell membrane permeabilizes and loses the expansion and retraction capacity and fixes cells, then a buffer solution (0 to 10 x PBS solution) is added to further cause the red blood cells to break due to the loss of the expansion and retraction capacity, and the main essence is that the extracellular osmotic pressure is changed from high to low by different solute salt solutions, so that the cells are changed from contraction to expansion, and the red blood cells break. The damage to non-erythrocyte caused by contraction and expansion in the process is also extremely serious, and the activity of the subsequently cultured and transplanted leucocyte is greatly influenced by permeabilized cell membranes and immobilized cells, and moreover, the distribution of antigen on the surface of the leucocyte is changed by the contraction and expansion, so that the flow type cell sorting is not facilitated. The application 201480060814.2 patent uses low concentrations of ammonium salts and HEPES and surfactants to lyse erythrocytes, requiring prolonged incubation during operation, and reports that ammonium salt-based erythrocyte lysis methods tend to be inadequate, leave more erythrocytes, affect subsequent flow sorting, and are also useful for cells with less cell abundance, such as CD34 + Hematopoietic stem cells are severely lost.
The main problems of the erythrocyte lysis reagent in the comprehensive current stage are as follows: first, most erythrocyte lysing agents depend on foreign importation, and the price is high and the goods picking period is long. Secondly, the complex operation and long cleavage reaction time lead the white blood cells to be exposed in a non-cell culture environment for a long time. Third, the red blood cells cannot be effectively protected while the red blood cells are rapidly and efficiently split in the reaction process, and residual red blood cell fragments cannot be thoroughly removed after the reaction is finished. In all documents and patents the problem of multi-cell debris remaining in the white blood cells collected by centrifugation after lysing the red blood cells is not solved, and the remaining cell debris directly affects the incubation of antibodies during flow sorting and the culture of the blocked flow sorting tubes and the transplanted cells. Fourth, the erythrocyte lysis reagent has poor compatibility with respect to various samples, such as inconsistent results when performing a split red reaction on alveolar lavage fluid, umbilical cord blood, whole blood, concentrated blood (plasma removed), bone marrow, spleen, etc., and the actual pH value of the reaction process is not easily stabilized.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a composition for rapidly lysing peripheral red blood cells and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the present invention provides a composition for the rapid lysis of peripheral red blood cells, the red blood cell lysate comprising one or more components selected from the group consisting of: glucose, calcium chloride, sodium chloride, potassium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, DIPSO (3- [ N-N-bis (2-hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid), betaine, meglumine diatrizoate, glycerol, and Tween 20;
the pH value of the composition is 7.0-7.6.
Further, the composition is composed of an aqueous solution of the following components: glucose, calcium chloride, sodium chloride, potassium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, DIPSO, betaine, meglumine diatrizoate, glycerol and Tween 20, and the final pH value is adjusted to 7.0-7.6.
Further, the composition comprises the following components in final concentration: glucose 2.0%, calcium chloride 6.0-8.0 mM/L, sodium chloride 50.0-57.0 mM/L, potassium chloride 0.8-1.2 mM/L, magnesium chloride 30.0-35.0 mM/L, potassium dihydrogen phosphate 5.0-5.5 mM/L, sodium dihydrogen phosphate 0.60-0.75 mM/L, DIPSO 15.0-20.0 mM/L, betaine 25.0-30.0 mM/L, meglumine diatrizoate 2.0-3.0%, glycerol 3.0-5.0% and Tween-20.5-1.0%.
Further, the pH of the above composition was 7.42.
In a second aspect, the invention provides the use of the above composition for the preparation of a red blood cell lysate.
In a third aspect, the invention provides the use of the above composition for lysing erythrocytes in blood, the volume ratio of whole blood to the above composition being 1 (4-8).
Further, the volume ratio of whole blood to the above composition was 1:6.
In a fourth aspect, the present invention provides a method of lysing erythrocytes in blood using the composition described above, comprising the steps of:
step one, adding whole blood into the composition according to the volume ratio of the whole blood to the composition of 1 (4-8), slowly reversing the upper part and the lower part, gently shaking, uniformly mixing, and centrifuging; and
and step two, removing the upper liquid, sucking and removing a whole yellow clot in the residual liquid, and cleaning the white blood cell mass at the bottom of the residual tube.
Further, the volume ratio of whole blood to the composition in the first step is 1:6.
Compared with the prior art, the invention has the following technical effects:
according to the invention, the hypotonic salt solution is adopted to rapidly deplete erythrocytes, so that erythrocytes are furthest lysed in the shortest time and the non-erythrocyte activity state is maintained, and in addition, erythrocyte fragments can be far away from cell clusters during centrifugation and enrichment of leukocytes, so that the purpose of removing the cell fragments is achieved; solves the problems of complicated and long time consumption, insufficient cracking and non-erythrocyte activity reduction of the prior red cracking reagent.
Drawings
FIG. 1 shows inverted microscopic images of leukocytes obtained at various time points after treatment of peripheral blood of tumor patients with lysate 1 (panel A) and lysate 2 (panel B);
FIG. 2 shows the results of flow-sorting of white blood cells obtained after treatment of peripheral blood of a tumor patient with lysis solution 1 (FIG. A) and lysis solution 2 (FIG. B) in accordance with an embodiment of the present invention;
FIG. 3 shows inverted microscopic images of monocytes obtained after treatment of peripheral blood of tumor patients with lysate 3 at various time points;
FIG. 4 shows the results of mononuclear cell flow sorting obtained after treatment of peripheral blood of a tumor patient with lysis solution 3 in an embodiment of the invention;
FIG. 5 shows a culture state diagram (40X) of cells obtained by treating venous blood of a tumor patient with lysate 1 (FIG. A), control 1 (FIG. B), control 2 (FIG. C), and control 3 (FIG. D) according to an embodiment of the present invention.
Detailed Description
The invention provides a composition for rapid lysis of peripheral red blood cells, wherein the red blood cell lysate comprises one or more of the following components: glucose, calcium chloride, sodium chloride, potassium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, DIPSO, betaine, meglumine diatrizoate, glycerol, and tween 20; the pH value of the composition is 7.0-7.6. Wherein, calcium chloride, sodium chloride, potassium chloride and magnesium chloride are basic ions for maintaining normal activity of cells and communicating and transmitting substances in and out of cells, and the basic ions play an important role in maintaining cell water balance, osmotic balance and ion balance; potassium dihydrogen phosphate and sodium dihydrogen phosphate are important substances for maintaining the balance of the pH value and isoelectric point inside and outside cells; betaine is a quaternary ammonium salt amphoteric surfactant, and has the effects of regulating cell osmotic pressure, relieving stimulation, and stabilizing the activity of cell surface proteins; tween-20 can protect cell surface antigen antibody protein, can reduce antibody damage to antigen, so as to carry out flow cell sorting subsequently; the diatrizoic acid meglumine can thicken the erythrocyte lysate and increase the viscosity of the liquid, so that erythrocyte membranes or cell fragments thereof can obtain larger centrifugal resistance without gathering the erythrocyte membranes and the non-erythrocyte fragments when centrifugally collecting the non-erythrocyte, thereby achieving the purpose of separating the cell fragments.
In a preferred embodiment of the invention, the above composition consists of an aqueous solution of: glucose, calcium chloride, sodium chloride, potassium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, DIPSO, betaine, meglumine diatrizoate, glycerol, and tween 20; the final pH was adjusted to 7.0-7.6.
In a preferred embodiment of the invention, the above composition comprises the following components in the final concentration: 2.0 to 3.0 percent of glucose, 6.0 to 8.0mM/L of calcium chloride, 50.0 to 57.0mM/L of sodium chloride, 0.8 to 1.2mM/L of potassium chloride, 30.0 to 35.0mM/L of magnesium chloride, 5.0 to 5.5mM/L of monopotassium phosphate, 0.60 to 0.75mM/L of sodium dihydrogen phosphate, 15.0 to 20.0mM/L of DIPSO,25.0 to 30.0mM/L of betaine, 2.0 to 3.0 percent of meglumine diatrizoate, 3.0 to 5.0 percent of glycerol and 0.5 to 1.0 percent of tween-20. Optimally, the composition comprises the following components in final concentration: glucose 2.0%, calcium chloride 7.10mM/L, sodium chloride 53.5mM/L, potassium chloride 0.9mM/L, magnesium chloride 33.5mM/L, potassium dihydrogen phosphate 5.3mM/L, sodium dihydrogen phosphate 0.71mM/L, DIPSO15.0 mM/L, betaine 27.5mM/L, meglumine diatrizoate 2.5%, glycerol 2.3%, tween-20.7%.
In a preferred embodiment of the invention, the pH of the above composition is 7.42.
The present invention also provides a method for lysing red blood cells in blood using the above composition as a lysing agent, in a preferred embodiment of the invention, the method comprises the steps of:
1. adding anticoagulated whole blood in a 50ml centrifuge tube according to the volume ratio of 1:5 (whole blood: red cracking reagent), slowly reversing the top and bottom, and mixing uniformly;
2. centrifugation at 3500rpm for 5 min at room temperature;
3. directly pouring out the upper liquid, taking a whole piece of yellow clot in the residual liquid as a cell fragment group, sucking out the cell fragment group by using a 1ml gun head, and taking the white cell group at the bottom of the residual tube as white cells.
4. White blood cells were washed by adding 1 XPBS.
By adopting the hypotonic salt solution and the method, the invention can crack red blood cells to the maximum extent and keep the non-red blood cell activity state.
The present invention will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.
The methods described in the examples are carried out using conventional methods, if not specified, and the reagents used are, if not specified, conventional commercially available reagents or reagents formulated by conventional methods.
Example 1
This example provides a composition for the rapid lysis of peripheral red blood cells comprising the following final concentrations of the components: 2.0% glucose, 6.0mM/L calcium chloride, 50.0mM/L sodium chloride, 0.8mM/L potassium chloride, 30.0mM/L magnesium chloride, 5.0mM/L potassium dihydrogen phosphate, 0.60mM/L sodium dihydrogen phosphate, 15.0mM/LDIPSO,25.0mM/L betaine, 2.0% meglumine diatrizoate, 1.5% glycerol, 0.5% Tween 20; the final pH of the composition was 7.42.
Example 2
This example provides a composition for the rapid lysis of peripheral red blood cells comprising the following final concentrations of the components: 2.0% glucose; 8.0mM/L calcium chloride; 57.0mM/L sodium chloride; 1.2mM/L potassium chloride; 35.0mM/L magnesium chloride; 5.5mM/L potassium dihydrogen phosphate; 0.75mM/L sodium dihydrogen phosphate; 15.0mM/LDIPSO;30.0mM/L betaine; meglumine diatrizoate 3.0%; 3.0% glycerol; 1.0% tween 20; the final pH of the composition was 7.42.
Example 3
This example provides a composition for the rapid lysis of peripheral red blood cells comprising the following final concentrations of the components: 2.0% glucose, 7.10mM/L calcium chloride, 53.5mM/L sodium chloride, 0.9mM/L potassium chloride, 33.5mM/L magnesium chloride, 5.3mM/L potassium dihydrogen phosphate, 0.71mM/L sodium dihydrogen phosphate, 15.0mM/LDIPSO,27.5mM/L betaine, 2.5% meglumine diatrizoate, 2.3% glycerol, 0.7% Tween 20; the final pH of the composition was 7.42.
Verification example 1
In this example, the composition provided in the above examples 1 and 2 was used as a lysate (named lysate 1 and lysate 2, respectively) to treat peripheral blood of tumor patients to obtain leukocytes and perform flow sorting, and specific operation steps and results are as follows:
1.5 ml of venous blood of a tumor patient is taken by using a 5ml EDTA-K2 anticoagulation tube, added into 2 50ml centrifuge tubes, and then 30ml of lysate 1 and lysate 2 are respectively added;
2. slowly reversing the centrifugal tube up and down, gently shaking, uniformly mixing, centrifuging at 23 ℃ and 3500rpm for 5 minutes, and then directly pouring out the upper liquid and sucking out cell debris clusters;
3. the reaction was stopped by adding 5ml of 1 XPBS, gently resuspension and washing the cells with a pipette, and then centrifuging at 23℃at 3500rpm for 5 minutes;
4. the PBS was discarded, 1ml of 1 XPBS was added again to resuspend the cells, and the cell suspension was split into two parts, one for cell culture and the other for flow cell sorting;
5. adding one part of cells into a 6-well plate containing 1640 culture medium, culturing in a 37 ℃ cell culture box, and observing cell states at 2h, 12h, 24h, 48h and 72h respectively, wherein the culture result is shown in figure 1;
6. another cell fraction was subjected to flow sorting at 4 ℃,500G, centrifugation for 5 min, supernatant removed, then 500 μl of 1 x cell staining solution (BioLegend, cat.No.420201) was added to resuspend the cells, followed by centrifugation at 4 ℃,500G for 5 min, and supernatant removed;
7. 100 μ l Fc Receptor Blocking Solution (BioLegend, cat.No.422301) was added and reacted at room temperature for 15 minutes, then a pre-formulated antibody solution was added and incubated at room temperature for 30 minutes; the antibodies used were BioLegend human peripheral blood cell flow sorting antibodies:
a.FITC anti-human CD11b Antibody；
b.Alexa Flour 700antibody CD3；
c.PerCP anti-human CD45 Antibody；
d.APC/Cy7 anti-human CD45 Antibody；
e.AP/cy7 anti-human CD8a Antibody；
f.APC anti-human CD56 Antibody HIB19；
g.PE anti-human CD56 Antibody；
8. 1ml of 1 Xcell staining solution was added and gently mixed, followed by centrifugation at 500G for 5 minutes at room temperature;
9. add 500. Mu.l of 1 XTrue-Nuclear TM The solution was reacted at room temperature for 60 minutes in the dark, then 1ml True-Nuclear was added TM 1 XPerm buffer, centrifuging at room temperature of 500G for 5 min, and discarding supernatant;
10. adding True-Nuclear TM 1ml of 1 XPerm buffer, centrifuging at room temperature of 500G for 5 minutes, and discarding the supernatant;
11. adding a pre-configured Violet421TM anti-human FOXP3 Antibody (320124, bioleged) Antibody, and incubating for 30 minutes at room temperature in the absence of light;
12. adding True-Nuclear TM 1ml of 1 XPerm buffer, centrifuging at room temperature of 500G for 5 minutes, and discarding the supernatant;
13. adding 1ml of 1 Xcell staining solution to resuspend cells, centrifuging at room temperature and 500G for 5 minutes, and removing the supernatant;
14. adding 300 μl of 1×cell staining solution to resuspend cells to perform flow separation of white blood cells; the results are shown in FIG. 2.
Verification example 2
In this example, the composition provided in the above example 3 was used as a lysate (named lysate 3) to treat peripheral blood of tumor patients to obtain mononuclear cells, and flow sorting was performed, and specific operation steps and results were as follows:
1.5 ml venous blood from tumor patients was collected using 5ml EDTA-K2 anticoagulant tube and centrifuged at 3000rpm at 4℃for 10 minutes. The blood in the anticoagulation tube is clearly seen to be divided into three layers, wherein the uppermost transparent yellow liquid is blood plasma, the middle white layer is mainly white blood cells, and the bottom red is red blood cells and other cells.
2. Sucking the upper serum and the middle white blood cells by a 1ml gun head, and then sucking 1-2ml of red blood cells from the top of the bottom red blood cells;
3. the bottom of the red blood cells is gently penetrated into the wall of the blood collection tube by a 1ml gun head, 500 mu l of tube bottom cells are gently sucked, the tube bottom cells are added into 5ml of lysate 3, the centrifuge tube is slowly reversed from top to bottom, the cells are gently mixed down, the room temperature is 3500rpm, the centrifugation is carried out for 5 minutes, and then the upper layer liquid is directly discarded and the cell debris clusters are sucked.
4. The PBS was discarded, 1ml of 1 XPBS was added again to resuspend the cells, and the cell suspension was split into two parts, one for cell culture and the other for flow cell sorting.
5. One cell was added to a 6-well plate containing 1640 medium, cultured in a 37℃cell incubator, and the cell states were observed at 2h, 12h, 24h, 48h, and 72h, respectively, and the results are shown in FIG. 3.
6. Another cell fraction was subjected to flow-sorting at 4℃for 500G, centrifuged for 5 minutes to remove the supernatant, and then 500. Mu.l of 1 Xcell staining solution (BioLegend, cat.No.420201) was added to resuspend the cells, followed by centrifugation at 4℃for 5 minutes to remove the supernatant.
7. 100 μ l Fc Receptor Blocking Solution (BioLegend, cat.No.422301) was added and reacted at room temperature for 15 minutes, then a pre-formulated antibody solution was added and incubated at room temperature for 30 minutes. The antibodies used were BioLegend human peripheral blood cell flow sorting antibodies:
a.FITC anti-human CD11b Antibody；
b.Alexa Flour 700antibody CD3；
c.PerCP anti-human CD45 Antibody；
d.APC/Cy7 anti-human CD45 Antibody；
e.AP/cy7 anti-human CD8a Antibody；
f.APC anti-human CD56 Antibody HIB19；
g.PE anti-human CD56 Antibody。
8. 1ml of 1X cell staining buffer was added and gently mixed, followed by centrifugation at 500G for 5 minutes at room temperature.
9. Add 500. Mu.l of 1 XTrue-Nuclear TM The solution was reacted at room temperature for 60 minutes in the dark, then 1ml True-Nuclear was added TM 1 XPerm buffer was mixed, centrifuged at 500G for 5 minutes at room temperature, and the supernatant was discarded.
10. Adding True-Nuclear TM 1ml of 1 XPerm buffer was mixed and centrifuged at 500G for 5 minutes at room temperature, and the supernatant was discarded.
11. Adding a preconfigured Violet421 TM anti-human FOXP3 anti-body (320124, bioleged) Antibody was incubated for 30 min at room temperature in the absence of light.
12. Adding True-Nuclear TM 1ml of 1 XPerm buffer was mixed and centrifuged at 500G for 5 minutes at room temperature, and the supernatant was discarded.
13. 1ml of 1X cell staining buffer cells were added and resuspended, after which the cells were centrifuged at 500G for 5 minutes at room temperature and the supernatant removed.
14. The flow-through separation of leukocytes was performed by adding 300. Mu.l of 1 Xcell staining solution to resuspend the cells, and the results are shown in FIG. 4.
Verification example 3
In this example, three control groups were set, and the effect of the lysate 3 provided in example 3 was verified, and specific operation steps and results were as follows:
control group 1:2.0% glucose, 15.0mM/L DIPSO,27.5mM/L betaine, 2.5% meglumine diatrizoate, 2.3% glycerol, 0.7% Tween 20, pH of the lysate was 7.42.
Control group 2:2.0% glucose, 7.10mM/L calcium chloride, 53.5mM/L sodium chloride, 0.9mM/L potassium chloride, 33.5mM/L magnesium chloride, 5.3mM/L potassium dihydrogen phosphate, 0.71mM/L sodium dihydrogen phosphate, 15.0mM/LDIPSO,2.5% meglumine diatrizoate, 2.3% glycerol, and pH of the lysate was 7.42.
Control group 3:2.0% glucose, 7.10mM/L calcium chloride, 53.5mM/L sodium chloride, 0.9mM/L potassium chloride, 33.5mM/L magnesium chloride, 5.3mM/L potassium dihydrogen phosphate, 0.71mM/L sodium dihydrogen phosphate, 15.0mM/LDIPSO,27.5mM/L betaine, 2.3% glycerol, 0.7% Tween 20, and pH of the lysate of 7.42.
Experimental procedure and results:
1.5 ml venous blood of a tumor patient is taken by using 5ml EDTA-K2 anticoagulant tube, added into a 50ml centrifuge tube, and then 30ml lysate 1, control group 2 and control group 3 are respectively added.
2. The centrifuge tube was slowly turned upside down and gently shaken down to mix well, centrifuged at 23℃at 3500rpm for 5 minutes, and then the supernatant liquid was directly discarded and the cell debris mass was sucked off.
3. The reaction was stopped by adding 5ml of 1 XPBS, gently resuspension and washing the cells with a pipette, and then centrifuging at 23℃at 3500rpm for 5 minutes.
4. The PBS was discarded, 500. Mu.l of 1 XPBS was added again to resuspend the cells, the cells were added to a 6-well plate containing 1640 medium, cultured in a 37℃cell incubator, and the cell state was observed after standing for 3 hours, and the results are shown in FIG. 5.
As can be seen from FIG. 5, the cells treated with lysate 1 are normal, the dead cells are small, and the outlines of the cells under the microscope are clear; the control group 1 has poor cell state, clustered cells, increased bright cells under a microscope and more dead cells; the control group 2 has normal cell state, small dead cell number, few red blood cells and clear cell outline under a microscope; control group 3 had normal cell status, few dead cells, more cell debris, and few erythrocytes.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (7)
1. A composition for rapid lysis of peripheral red blood cells comprising the following final concentrations of components: glucose 2.0%, calcium chloride 6.0-8.0 mM/L, sodium chloride 50.0-57.0 mM/L, potassium chloride 0.8-1.2 mM/L, magnesium chloride 30.0-35.0 mM/L, potassium dihydrogen phosphate 5.0-5.5 mM/L, sodium dihydrogen phosphate 0.60-0.75 mM/L, DIPSO 15.0-20.0 mM/L, betaine 25.0-30.0 mM/L, meglumine diatrizoate 2.0-3.0%, glycerol 3.0-5.0%, tween-20.5-1.0%;
the pH value of the composition is 7.0-7.6.
2. The composition of claim 1, wherein the pH of the composition is 7.42.
3. Use of a composition according to any one of claims 1-2 for the preparation of a red blood cell lysate.
4. Use of a composition according to any of claims 1-2 for lysing erythrocytes in blood, wherein the volume ratio of whole blood to the composition is 1 (4-8).
5. The use according to claim 4, wherein the volume ratio of whole blood to the composition is 1:6.
6. A method of lysing erythrocytes in blood using a composition according to any of claims 1 to 2, comprising the steps of:
step one, adding whole blood into the composition according to the volume ratio of the whole blood to the composition of 1 (4-8), slowly reversing the upper part and the lower part, gently shaking, uniformly mixing, and centrifuging; and
and step two, removing the upper liquid, sucking and removing a whole yellow clot in the residual liquid, and cleaning the white blood cell mass at the bottom of the residual tube.
7. The method of claim 6, wherein the volume ratio of whole blood to the composition in step one is 1:6.
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| CN103627638A (en) * | 2013-05-10 | 2014-03-12 | 北京东方华辉生物医药科技有限公司 | Composition for lysing red cells, red cell lysing reagent and application of red cell lysing reagent |
| CN103789298A (en) * | 2013-12-30 | 2014-05-14 | 湖南圣湘生物科技有限公司 | Erythrocyte lysis buffer and application thereof |
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| CN102747036A (en) * | 2011-04-19 | 2012-10-24 | 北京同盛颐和生物科技有限公司 | Kit for in-vitro separation purification of karyocytes and removal of erythrocytes and use method of the kit |
| CN103627638A (en) * | 2013-05-10 | 2014-03-12 | 北京东方华辉生物医药科技有限公司 | Composition for lysing red cells, red cell lysing reagent and application of red cell lysing reagent |
| CN103789298A (en) * | 2013-12-30 | 2014-05-14 | 湖南圣湘生物科技有限公司 | Erythrocyte lysis buffer and application thereof |
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