CN106771117A - A kind of preparation method of negative film of antigen of neutrophilic granulocyte - Google Patents
A kind of preparation method of negative film of antigen of neutrophilic granulocyte Download PDFInfo
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- CN106771117A CN106771117A CN201611051867.5A CN201611051867A CN106771117A CN 106771117 A CN106771117 A CN 106771117A CN 201611051867 A CN201611051867 A CN 201611051867A CN 106771117 A CN106771117 A CN 106771117A
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Abstract
The invention discloses a kind of preparation method of negative film of antigen of neutrophilic granulocyte, comprise the following steps：By the heparin anti-coagulating of people with sodium chloride solution be well mixed, room temperature place, draw upper plasma；By in upper plasma human lymphocyte separating liquid of the addition containing DextranT500, fully mix, centrifugation, absorption is deposited in the leukocytic cream solution of bottom, obtains solution to be centrifuged；Added described in centrifuge tube after centrifugation solution mixes with LymphoprepTM separating liquids, centrifuge tube is placed in horizontal type centrifuger and is centrifuged；Neutrophil leucocyte layer solution is drawn, cell suspending liquid is prepared；Cell suspending liquid is added dropwise on clean slide, natural air drying, last absolute ethyl alcohol is fixed, and obtains negative film of antigen of neutrophilic granulocyte.Preparation method of the invention, is solved in the existing method for preparing negative film of antigen of neutrophilic granulocyte, there are problems that neutrophil leucocyte is pockety, and reaction result is easily observed.
Description
Technical field
The invention belongs to negative film of antigen preparing technical field, and in particular to a kind of preparation side of negative film of antigen of neutrophilic granulocyte
Method.
Background technology
ANCA (anti-neutrophil cytoplasmic antibody, ANCA), refer to
The antibody that lysosomal enzyme reacts in neutrophil leucocyte and monocyte endochylema.It is many to systemic vasculitis IBD etc.
The diagnosis and differential diagnosis for planting disease have great importance, it has also become a very important routine of autoimmune disease
Detection project.
Indirect immuno fluorescent method is to detect reference method or the standard method of ANCA, is so far also the most frequently used classics
Method.Generally need to prepare the negative film of antigen of neutrophilic granulocyte that can be reacted before the method detection ANCA, work as ANCA
After being reacted with neutrophil leucocyte, then result observation can be carried out.
In the prior art, the neutrophil leucocyte fixed using ethanol is one of the most frequently used negative film of antigen preparation method, first
The neutrophil leucocyte that will first isolate is coated on slide, is then fixed by ethanol, formation negative film of antigen, but the method
The negative film of antigen of preparation has that cell distribution is uneven, so that, after neutrophil leucocyte and antibody response, it is difficult observation
Reaction result, influences the observation of ANCA testing results to judge.
The content of the invention
A kind of preparation method of negative film of antigen of neutrophilic granulocyte that the present invention is provided, solves and existing prepares neutrophil leucocyte
In the method for negative film of antigen, there are problems that neutrophil leucocyte is pockety, reaction result is easily observed.
A kind of preparation method of negative film of antigen of neutrophilic granulocyte that the present invention is provided, comprises the following steps：
Step 1, by the sodium chloride solution of the heparin anti-coagulating of people and 0.5g/100g according to 4~6:1 volume ratio mixing
Uniformly, room temperature is placed, until erythroprecipitin, draws upper plasma, it is standby；
Step 2, by upper plasma human lymphocyte separating liquid of the addition containing DextranT500, fully mixes, then
Room temperature, 3000r/min centrifugation 10-15min, absorption are deposited in the leukocytic cream solution of bottom, obtain solution to be centrifuged；
Wherein, the DextranT500 of 1.5g is added in every 100mL human lymphocyte separating liquids；
Step 3, by the solution to be centrifuged and LymphoprepTM separating liquids by 1:After 3~5 volume ratio mixing, plus
Enter in centrifuge tube, centrifuge tube is placed in horizontal type centrifuger, room temperature, 1000-2000r/min centrifugations 30-35min；
Step 4, draws neutrophil leucocyte layer solution, adds 4 DEG C of buffer solutions of precooling, 4 DEG C, 1000r/min centrifugations 5-
10min, abandons supernatant, collects cell precipitation；
It is prepared from the following components in every liter of buffer solution：The sodium chloride of 5g, the potassium chloride of 3g, distilled water are settled to
1L；
Step 5, to 4 DEG C of 0.8-0.9g/100g sodium chloride solutions of precooling are added in cell precipitation, make cell number for 5~
5.5×105ML, obtains cell suspending liquid；
Step 6, cell suspending liquid is added dropwise on clean slide, natural air drying, and the anhydrous of 4 DEG C of precoolings is finally added dropwise
Ethanol, fixed 5-8min, obtains negative film of antigen of neutrophilic granulocyte after natural air drying.
Preferably, in the preparation method of above-mentioned negative film of antigen of neutrophilic granulocyte, in step 1, the time that room temperature is placed is 30-
40min。
Preferably, in the preparation method of above-mentioned negative film of antigen of neutrophilic granulocyte, the structure of the centrifuge tube includes：Reception pipe
With the screen pipe being sheathed in reception pipe, screen pack is laterally fixed with the screen pipe, the mesh diameter of the screen pack is
0.4-0.6 μm, annular stop plate is provided with around the outer wall of the screen pipe, and the annular stop plate is covered in the reception
On the mouth of pipe of pipe, and the annular stop plate and the reception pipe the mouth of pipe by seal with elastometic washer, the screen pipe it is upper
End is provided with the lid for covering screen pipe upper orifice, and the lower end of the screen pipe offers lower nozzle, under the screen pipe
Certain distance is left between the mouth of pipe and the reception pipe inner bottom part.
Preferably, in the preparation method of above-mentioned negative film of antigen of neutrophilic granulocyte, step 2 also includes to red blood cell in step 1
The collection of the neutrophil leucocyte in precipitation, specially：
Precipitated with the sodium chloride solution lysed erythrocyte of 4 DEG C, 2g/100g, the PBS for adding 0.01mol/L, pH to be 7.0 fills
Divide and 5-10min is stood after mixing, then 4 DEG C, 1000r/min centrifugation 5-10min, collect supernatant；By supernatant and leukocytic cream
Solution mixes, and obtains solution to be centrifuged.
Compared with prior art, a kind of preparation method of negative film of antigen of neutrophilic granulocyte that the present invention is provided has and following has
Beneficial effect：
(1) solve in the existing method for preparing negative film of antigen of neutrophilic granulocyte, there is neutrophil leucocyte skewness
Problem so that, when neutrophil leucocyte and after antibody response, reaction result is easily observed, it is ensured that the reliability of ANCA testing results
Property.
(2) using the sodium chloride solution treatment heparin anti-coagulating of 0.5g/100g, neutrophil leucocyte physiologically active can be kept
And conformation of rules, while accelerating erythroprecipitin, 30-40min red blood cells can be precipitated completely.
(3) neutrophil leucocyte is separated using LymphoprepTM separating liquids, separating effect is obvious, and operation side
Just, and the operation that is centrifuged again after centrifugation solution mixes with LymphoprepTM separating liquids, rather than conventional centrifugal are used
Bottom of the tube puts LymphoprepTM separating liquids, and solution to be centrifuged is put on LymphoprepTM separating liquids upper strata, during centrifugation,
LymphoprepTM separating liquids are fully contacted with solution to be centrifuged, and improve separative efficiency, and cell is dense in increasing neutrophil leucocyte layer
Degree；
The centrifuge tube used in preparation method of the invention is the centrifuge tube of special construction, is set in the screen pipe of centrifuge tube
Have a screen pack, and screen pack mesh diameter at 0.4-0.6 μm, when the material of also Multiple components passes through the screen pack,
Peptizaiton is played, heterogeneity is uniformly acted on LymphoprepTM separating liquids, so as to increase separative efficiency；Separately
Outward, due to this mesh close to the particle diameter of neutrophil leucocyte so that by centrifugation, neutrophil leucocyte is dispersed, in order to
When subsequently preparing egative film, cell has preferable decentralization, it is ensured that cell is uniformly dispersed on slide, is easy to observation.This
Outward, after using the centrifuge tube, the high purity more than 95% of the neutrophil leucocyte isolated.
(4) sodium chloride solution is added in step 4 using buffer solution for cleaning neutrophil leucocyte layer solution, in step 5, can be with
The physiologically active of neutrophil leucocyte is protected not suffer a loss, the influence by neutrophils antigen substrate to ANCA testing results is dropped to
It is minimum.
Brief description of the drawings
Fig. 1 is the structural representation of centrifuge tube in the present invention.
In figure, 1. reception pipe, 2. screen pipe, 21. screen packs, 22. annular stop plates, 23. lids.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail, but should not be construed as limitation of the invention.It is following
The test method of unreceipted actual conditions in embodiment, generally operates according to normal condition, due to not being related to inventive point, thus it is not right
Its step is described in detail.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can select between point and two end points.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to prior art can also be used and this
Any method, equipment and material in the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
A kind of preparation method of negative film of antigen of neutrophilic granulocyte that the present invention is provided, comprises the following steps：
Step 1, by the sodium chloride solution of the heparin anti-coagulating of people and 0.5g/100g according to 4~6:1 volume ratio mixing
Uniformly, room temperature places 30-40min, until erythroprecipitin, draws upper plasma, it is standby；It should be noted that the heparin is anti-
Blood coagulation is preferably with fresh heparin anti-coagulating.
Step 2, by upper plasma add containing DextranT500 (Dextran T 500) human lymphocyte separating liquid in,
Fully mix, then room temperature, 3000r/min centrifugation 10-15min, absorption is deposited in the leukocytic cream solution of bottom, obtain treating from
Heart solution；Wherein, the DextranT500 of 1.5g is added in every 100mL human lymphocyte separating liquids, wherein, human lymphocyte point
Chaotropic is purchased from Hefei Bo Mei biotechnologies Co., Ltd.
Step 3, by the solution to be centrifuged and LymphoprepTM separating liquids by 1:After 3~5 volume ratio mixing, plus
Enter in centrifuge tube, centrifuge tube is placed in horizontal type centrifuger, room temperature, 1000-2000r/min centrifugations 30-35min；After centrifugation
Neutrophil leucocyte layer solution is located at the interface of bottom blood and LymphoprepTM separating liquids, and interface is more obvious；
The LymphoprepTM separating liquids are the LymphoprepTM separating liquids of Axis-Shield companies of Norway production.
The structure of the centrifuge tube is described as shown in figure 1, including reception pipe 1 and the screen pipe 2 being sheathed in reception pipe 1
Screen pack 21 is laterally fixed with screen pipe 2, the mesh diameter of the screen pack 21 is 0.4-0.6 μm, around the screen pipe 2
Outer wall be provided with annular stop plate 22, and annular stop plate 22 is covered on the mouth of pipe of the reception pipe 1, and annular stop
Plate 22 passes through seal with elastometic washer with the mouth of pipe of the reception pipe 1, and the upper end of the screen pipe 2 is provided with for covering screen pipe 2
The lid 23 of the mouth of pipe, the lower end of the screen pipe 2 offers lower nozzle, the lower nozzle of screen pipe 2 and the inner bottom part of the reception pipe 1
Between leave certain distance.
When the solution of addition is by after screen pack 21, due to screen pack 21 mesh diameter close to neutrophil leucocyte grain
Footpath so that by centrifugation, neutrophil leucocyte is dispersed, and during in order to subsequently preparing egative film, cell has preferable decentralization,
It is easy to observation.
All of solution enters in reception pipe 1 after centrifugation, and screen pipe 2 is taken out from reception pipe 1, draws in reception pipe 1
Neutrophil leucocyte layer solution, leave certain distance between the lower nozzle of screen pipe 2 and the bottom of the reception pipe 1, answer
When can suitably be adjusted according to actual needs, can be entered in reception pipe 1 with all of solution after ensureing centrifugation；Annular stop
Screen pipe 2 is fallen into reception pipe 1 completely when plate 22 is additionally operable to prevent centrifugation.
Step 4, draws neutrophil leucocyte layer solution, adds 4 DEG C of buffer solutions of precooling, 4 DEG C, 1000r/min centrifugations 5-
10min, abandons supernatant, collects cell precipitation；
It is prepared from the following components in every liter of buffer solution：The sodium chloride of 5g, the potassium chloride of 3g, distilled water are settled to
1L。
Step 5, to 4 DEG C of 0.8-0.9g/100g sodium chloride solutions of precooling are added in cell precipitation, make cell number for 5~
5.5×105ML, obtains cell suspending liquid.
Step 6, cell suspending liquid is added dropwise on clean slide, natural air drying, and the anhydrous of 4 DEG C of precoolings is finally added dropwise
Ethanol, fixed 5-8min, obtains negative film of antigen of neutrophilic granulocyte after natural air drying, -20 DEG C save backup.
It should be noted that in order to study all of neutrophil leucocyte in blood as far as possible, step 2 also includes to step
The collection of the neutrophil leucocyte in 1 erythroprecipitin, specially：
Precipitated with the sodium chloride solution lysed erythrocyte of 4 DEG C, 2g/100g, the PBS for adding 0.01mol/L, pH to be 7.0 fills
Divide and 5-10min is stood after mixing, then 4 DEG C, 1000r/min centrifugation 5-10min, collect supernatant；And by supernatant and leucocyte
Layer solution mixing, obtains solution to be centrifuged.
Then the centrifugation step of step 3 can be carried out, will solution be centrifuged and LymphoprepTM separating liquids by 1:3~
After 5 volume ratio mixing, add in centrifuge tube, then centrifuge tube is placed in horizontal type centrifuger, 1000-2000r/min,
Room temperature is centrifuged 30-35min；Neutrophil leucocyte layer solution is located at the interface of bottom blood and LymphoprepTM separating liquids after centrifugation
Place, and interface is more obvious.
Preferably, the preparation method of the negative film of antigen of neutrophilic granulocyte that the present invention is provided, including following examples：
Embodiment 1
A kind of preparation method of negative film of antigen of neutrophilic granulocyte, comprises the following steps：
Step 1, by the sodium chloride solution of the heparin anti-coagulating of people and 0.5g/100g according to 4:1 volume ratio mixing is equal
Even, room temperature places 30-40min, until erythroprecipitin, draws upper plasma, it is standby；
Step 2, by upper plasma human lymphocyte separating liquid of the addition containing DextranT500, fully mixes, then
Room temperature, 3000r/min centrifugation 10min, absorption are deposited in the leukocytic cream solution of bottom, obtain solution to be centrifuged；Wherein, often
The DextranT500 of 1.5g is added in 100mL human lymphocyte separating liquids, wherein, human lymphocyte separating liquid is rich purchased from Hefei
U.S. biotechnology Co., Ltd.
Precipitated with the sodium chloride solution lysed erythrocyte of 4 DEG C, 2g/100g, the PBS for adding 0.01mol/L, pH to be 7.0 fills
Divide and 5-10min is stood after mixing, then 4 DEG C, 1000r/min centrifugation 5-10min, collect supernatant；And by supernatant and leucocyte
Layer solution mixing, obtains solution to be centrifuged.
Step 3, by the solution to be centrifuged and LymphoprepTM separating liquids by 1:After 3 volume ratio mixing, add
In centrifuge tube, centrifuge tube is placed in horizontal type centrifuger, room temperature, 1000r/min centrifugations 30min；Neutrophil leucocyte after centrifugation
Layer solution is located at the interface of bottom blood and LymphoprepTM separating liquids, and interface is more obvious；
The LymphoprepTM separating liquids are the LymphoprepTM separating liquids of Axis-Shield companies of Norway production.
The structure of the centrifuge tube is described as shown in figure 1, including reception pipe 1 and the screen pipe 2 being sheathed in reception pipe 1
Laterally every screen pack 21 is fixed with screen pipe 2, the mesh diameter of the screen pack 21 is 0.4-0.6 μm, around the filtering
The outer wall of pipe 2 is provided with annular stop plate 22, and annular stop plate 22 is covered on the mouth of pipe of the reception pipe 1, and annular
Limiting plate 22 passes through seal with elastometic washer with the mouth of pipe of the reception pipe 1, and the upper end of the screen pipe 2 is provided with for covering screen pipe
The lid 23 of 2 upper orifices, the lower end of the screen pipe 2 offers lower nozzle, in the lower nozzle of screen pipe 2 and the reception pipe 1
Certain distance is left between bottom.
Step 4, absorption neutrophil leucocyte layer solution, 4 DEG C of buffer solutions of precooling of addition, 4 DEG C, 1000r/min centrifugation 5min,
Supernatant is abandoned, cell precipitation is collected；
It is prepared from the following components in every liter of buffer solution：The sodium chloride of 5g, the potassium chloride of 3g, distilled water are settled to
1L。
Step 5, to 4 DEG C of 0.8g/100g sodium chloride solutions of precooling are added in cell precipitation, make cell number for 5~5.5 ×
105ML, obtains cell suspending liquid.
Step 6, cell suspending liquid is added dropwise on clean slide, natural air drying, and the anhydrous of 4 DEG C of precoolings is finally added dropwise
Ethanol, fixed 5min, obtains negative film of antigen of neutrophilic granulocyte after natural air drying, -20 DEG C save backup.
Embodiment 2
A kind of preparation method of negative film of antigen of neutrophilic granulocyte, comprises the following steps：
Step 1, by the sodium chloride solution of the heparin anti-coagulating of people and 0.5g/100g according to 5:1 volume ratio mixing is equal
Even, room temperature places 30-40min, until erythroprecipitin, draws upper plasma, it is standby；
Step 2, by upper plasma human lymphocyte separating liquid of the addition containing DextranT500, fully mixes, then
Room temperature, 3000r/min centrifugation 12min, absorption are deposited in the leukocytic cream solution of bottom, obtain solution to be centrifuged；Wherein, often
The DextranT500 of 1.5g is added in 100mL human lymphocyte separating liquids, wherein, human lymphocyte separating liquid is rich purchased from Hefei
U.S. biotechnology Co., Ltd.
Step 3, by the solution to be centrifuged and LymphoprepTM separating liquids by 1:After 4 volume ratio mixing, add
In centrifuge tube, centrifuge tube is placed in horizontal type centrifuger, room temperature, 1500r/min centrifugations 32min；Neutrophil leucocyte after centrifugation
Layer solution is located at the interface of bottom blood and LymphoprepTM separating liquids, and interface is more obvious；
The LymphoprepTM separating liquids are the LymphoprepTM separating liquids of Axis-Shield companies of Norway production.
The structure of the centrifuge tube is with embodiment 1.
Step 4, absorption neutrophil leucocyte layer solution, 4 DEG C of buffer solutions of precooling of addition, 4 DEG C, 1000r/min centrifugation 8min,
Supernatant is abandoned, cell precipitation is collected；
It is prepared from the following components in every liter of buffer solution：The sodium chloride of 5g, the potassium chloride of 3g, distilled water are settled to
1L。
Step 5, to 4 DEG C of 0.85g/100g sodium chloride solutions of precooling are added in cell precipitation, makes cell number be 5~5.5
×105ML, obtains cell suspending liquid.
Step 6, cell suspending liquid is added dropwise on clean slide, natural air drying, and the anhydrous of 4 DEG C of precoolings is finally added dropwise
Ethanol, fixed 6min, obtains negative film of antigen of neutrophilic granulocyte after natural air drying, -20 DEG C save backup.
Embodiment 3
A kind of preparation method of negative film of antigen of neutrophilic granulocyte, comprises the following steps：
Step 1, by the sodium chloride solution of the heparin anti-coagulating of people and 0.5g/100g according to 6:1 volume ratio mixing is equal
Even, room temperature places 30-40min, until erythroprecipitin, draws upper plasma, it is standby；
Step 2, by upper plasma human lymphocyte separating liquid of the addition containing DextranT500, fully mixes, then
Room temperature, 3000r/min centrifugation 15min, absorption are deposited in the leukocytic cream solution of bottom, obtain solution to be centrifuged；Wherein, often
The DextranT500 of 1.5g is added in 100mL human lymphocyte separating liquids, wherein, human lymphocyte separating liquid is rich purchased from Hefei
U.S. biotechnology Co., Ltd.
Step 3, by the solution to be centrifuged and LymphoprepTM separating liquids by 1:After 5 volume ratio mixing, add
In centrifuge tube, centrifuge tube is placed in horizontal type centrifuger, room temperature, 2000r/min centrifugations 35min；Neutrophil leucocyte after centrifugation
Layer solution is located at the interface of bottom blood and LymphoprepTM separating liquids, and interface is more obvious；
The LymphoprepTM separating liquids are the LymphoprepTM separating liquids of Axis-Shield companies of Norway production.
The centrifuge tube is common centrifuge tube.
Step 4, draws neutrophil leucocyte layer solution, adds 4 DEG C of buffer solutions of precooling, 4 DEG C, 1000r/min centrifugations
10min, abandons supernatant, collects cell precipitation；
It is prepared from the following components in every liter of buffer solution：The sodium chloride of 5g, the potassium chloride of 3g, distilled water are settled to
1L。
Step 5, to 4 DEG C of 0.9g/100g sodium chloride solutions of precooling are added in cell precipitation, make cell number for 5~5.5 ×
105ML, obtains cell suspending liquid.
Step 6, cell suspending liquid is added dropwise on clean slide, natural air drying, and the anhydrous of 4 DEG C of precoolings is finally added dropwise
Ethanol, fixed 8min, obtains negative film of antigen of neutrophilic granulocyte after natural air drying, -20 DEG C save backup.
The negative film of antigen of neutrophilic granulocyte for being prepared in the method for embodiment 1 below, then using IIF pair
100 patients of systemic vasculitis carry out ANCA detections, wherein aorto-arteritis 20, rheumatic arthritis 60, systematicness
Lupus erythematosus 20 is as a result as follows：Wherein aorto-arteritis detection is positive 14, and positive rate is 70%；Rheumatic arthritis is detected
Positive 50, positive rate is 83.33%；Systemic loupus erythematosus detection is positive 10, and positive rate is 50%.
The blood of above-mentioned 100 patients is detected again using commercially available ANCA detection reagents, it is as a result as follows：It is wherein big
Arteritis detection is positive 8, and positive rate is 40%；Rheumatic arthritis detection is positive 40, and positive rate is 66.67%；System
Property positive 8 of lupus erythematosus detection, positive rate is 40%.
Illustrate, the antigen substrate being prepared using the method for the present invention can effectively improve neutrophil leucocyte in blood
Recall rate, significantly improves the recall rate of the patients such as aorto-arteritis, rheumatic arthritis, systemic loupus erythematosus, and effect is obvious.
It should be noted that the method for the present invention refers to the preparation of negative film of antigen, but the negative film of antigen is only to provide
One material, from the preparation method and cannot draw any disease detection result, and it is needed using existing method, with antibody
After reaction, can just there is testing result.
Further, we are respectively adopted the method and routine of embodiment 1 to be experimental subjects with portion blood sample
Ficoll-Hypaque density-gradient centrifugation methods collect neutrophil leucocyte layer solution, and neutrophil leucocyte in this layer of solution is detected respectively
Purity and survival rate, it is as a result as follows：
The purity of isolated neutrophil leucocyte is more than 95% in embodiment 1, and routine Ficoll-Hypaque density
The purity of the isolated neutrophil leucocyte of gradient centrifugation is more than 90%, illustrates that the method for the present invention can improve separation pure
Degree.
The survival rate of isolated neutrophil leucocyte is more than 99% in embodiment 1, and routine Ficoll-Hypaque is close
The survival rate of the isolated neutrophil leucocyte of degree gradient centrifugation is more than 90%, illustrates that the method for the present invention can improve neutrality
The survival rate of granulocyte, improves conventional efficient.
, but those skilled in the art once know basic creation although preferred embodiments of the present invention have been described
Property concept, then can make other change and modification to these embodiments.So, appended claims are intended to be construed to include excellent
Select embodiment and fall into having altered and changing for the scope of the invention.
Obviously, those skilled in the art can carry out various changes and modification without deviating from essence of the invention to the present invention
God and scope.So, if these modifications of the invention and modification belong to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising these changes and modification.
Claims (4)
1. a kind of preparation method of negative film of antigen of neutrophilic granulocyte, it is characterised in that comprise the following steps：
Step 1, by the sodium chloride solution of the heparin anti-coagulating of people and 0.5g/100g according to 4~6:1 volume ratio mixing is equal
Even, room temperature is placed, until erythroprecipitin, draws upper plasma, it is standby；
Step 2, by upper plasma human lymphocyte separating liquid of the addition containing Dextran T500, fully mixes, then room
Temperature, 3000r/min centrifugation 10-15min, absorption are deposited in the leukocytic cream solution of bottom, obtain solution to be centrifuged；
Wherein, the Dextran T500 of 1.5g are added in every 100mL human lymphocyte separating liquids；
Step 3, by the solution to be centrifuged and LymphoprepTM separating liquids by 1:3~5 volume ratio mixing after, add from
In heart pipe, centrifuge tube is placed in horizontal type centrifuger, room temperature, 1000-2000r/min centrifugations 30-35min；
Step 4, absorption neutrophil leucocyte layer solution, 4 DEG C of buffer solutions of precooling of addition, 4 DEG C, 1000r/min centrifugation 5-10min,
Supernatant is abandoned, cell precipitation is collected；
It is prepared from the following components in every liter of buffer solution：The sodium chloride of 5g, the potassium chloride of 3g, distilled water are settled to 1L；
Step 5, to 4 DEG C of 0.8-0.9g/100g sodium chloride solutions of precooling are added in cell precipitation, make cell number for 5~5.5 ×
105ML, obtains cell suspending liquid；
Step 6, cell suspending liquid is added dropwise on clean slide, natural air drying, and 4 DEG C of anhydrous second of precooling are finally added dropwise
Alcohol, fixed 5-8min, obtains negative film of antigen of neutrophilic granulocyte after natural air drying.
2. the preparation method of negative film of antigen of neutrophilic granulocyte according to claim 1, it is characterised in that in step 1, room temperature
The time of placement is 30-40min.
3. the preparation method of the negative film of antigen of neutrophilic granulocyte according to right wants 1, it is characterised in that the knot of the centrifuge tube
Structure includes：Reception pipe (1) and the screen pipe (2) being sheathed in reception pipe (1), filtering is laterally fixed with the screen pipe (2)
Net (21), the mesh diameter of the screen pack (21) is 0.4-0.6 μm, and the outer wall around the screen pipe (2) is provided with annular limit
Position plate (22), and the annular stop plate (22) covered on the mouth of pipe of the reception pipe (1), and the annular stop plate
(22) mouth of pipe with the reception pipe (1) passes through seal with elastometic washer, and the upper end of the screen pipe (2) is provided with for covering screen pipe
(2) lid (23) of upper orifice, the lower end of the screen pipe (2) offers lower nozzle, the lower nozzle of the screen pipe (2) and institute
State and leave certain distance between reception pipe (1) inner bottom part.
4. the preparation method of negative film of antigen of neutrophilic granulocyte according to claim 1, it is characterised in that step 2 also includes
Collection to the neutrophil leucocyte in the erythroprecipitin of step 1, specially：
Precipitated with the sodium chloride solution lysed erythrocyte of 4 DEG C, 2g/100g, add the PBS that 0.01mol/L, pH are 7.0, it is fully mixed
5-10min, then 4 DEG C, 1000r/min centrifugation 5-10min are stood after even, supernatant is collected；And supernatant is molten with leukocytic cream
Liquid mixes, and obtains solution to be centrifuged.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611051867.5A CN106771117B (en) | 2016-11-25 | 2016-11-25 | A kind of preparation method of negative film of antigen of neutrophilic granulocyte |
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| CN113186161A (en) * | 2021-03-24 | 2021-07-30 | 南京医科大学 | Method for quickly separating ultra-high purity human blood neutrophils |
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| US6027922A (en) * | 1993-11-02 | 2000-02-22 | Innogenetics N.V. | Human foam cells and methods for preparing them, monoclonal antibodies to said foam cells and their pharmaceutical and diagnostic use |
| CN1687780A (en) * | 2005-05-01 | 2005-10-26 | 李小峰 | Method for preparing negative film of antigen of neutrophilic granulocyte |
| CN102586187A (en) * | 2012-02-23 | 2012-07-18 | 深圳市中美康士生物科技有限公司 | In vitro preservation method and culture medium for neutrophils |
| CN105073725A (en) * | 2012-08-17 | 2015-11-18 | 瑞斯比维特有限公司 | Pyrazolyl-ureas as kinase inhibitors |
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| US6027922A (en) * | 1993-11-02 | 2000-02-22 | Innogenetics N.V. | Human foam cells and methods for preparing them, monoclonal antibodies to said foam cells and their pharmaceutical and diagnostic use |
| CN1687780A (en) * | 2005-05-01 | 2005-10-26 | 李小峰 | Method for preparing negative film of antigen of neutrophilic granulocyte |
| CN102586187A (en) * | 2012-02-23 | 2012-07-18 | 深圳市中美康士生物科技有限公司 | In vitro preservation method and culture medium for neutrophils |
| CN105073725A (en) * | 2012-08-17 | 2015-11-18 | 瑞斯比维特有限公司 | Pyrazolyl-ureas as kinase inhibitors |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113186161A (en) * | 2021-03-24 | 2021-07-30 | 南京医科大学 | Method for quickly separating ultra-high purity human blood neutrophils |
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