CN103740811B - Chromosome karyotype analysis marrow G is with preparation method - Google Patents
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- CN103740811B CN103740811B CN201310592287.7A CN201310592287A CN103740811B CN 103740811 B CN103740811 B CN 103740811B CN 201310592287 A CN201310592287 A CN 201310592287A CN 103740811 B CN103740811 B CN 103740811B
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- 210000000349 chromosome Anatomy 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 238000004458 analytical method Methods 0.000 title claims abstract description 7
- NNJPGOLRFBJNIW-HNNXBMFYSA-N (-)-demecolcine Chemical compound C1=C(OC)C(=O)C=C2[C@@H](NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-HNNXBMFYSA-N 0.000 claims abstract description 25
- NNJPGOLRFBJNIW-UHFFFAOYSA-N Demecolcine Natural products C1=C(OC)C(=O)C=C2C(NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229960005052 demecolcine Drugs 0.000 claims abstract description 25
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229960005542 ethidium bromide Drugs 0.000 claims abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims abstract description 6
- 210000002798 bone marrow cell Anatomy 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 10
- 238000004043 dyeing Methods 0.000 claims description 9
- 238000004113 cell culture Methods 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 8
- 239000011550 stock solution Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000000975 dye Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 239000006285 cell suspension Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000012224 working solution Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000006143 cell culture medium Substances 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000011435 rock Substances 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- 230000032683 aging Effects 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 108010019160 Pancreatin Proteins 0.000 claims description 2
- 238000013019 agitation Methods 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- 239000012153 distilled water Substances 0.000 claims description 2
- 239000012154 double-distilled water Substances 0.000 claims description 2
- 229940055695 pancreatin Drugs 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 102000004142 Trypsin Human genes 0.000 claims 1
- 108090000631 Trypsin Proteins 0.000 claims 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims 1
- 229910052782 aluminium Inorganic materials 0.000 claims 1
- 239000004411 aluminium Substances 0.000 claims 1
- 239000012588 trypsin Substances 0.000 claims 1
- 230000002759 chromosomal effect Effects 0.000 abstract description 4
- 239000000872 buffer Substances 0.000 description 9
- 238000000386 microscopy Methods 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002559 cytogenic effect Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000009671 shengli Substances 0.000 description 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- HOWHQWFXSLOJEF-MGZLOUMQSA-N systemin Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)OC(=O)[C@@H]1CCCN1C(=O)[C@H]1N(C(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H]2N(CCC2)C(=O)[C@H]2N(CCC2)C(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)C(C)C)CCC1 HOWHQWFXSLOJEF-MGZLOUMQSA-N 0.000 description 1
- 108010050014 systemin Proteins 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Abstract
The invention discloses in a kind of field of medical examination and be with preparation method for chromosome karyotype analysis marrow G, be included in while stopping cultivating, ethidium bromide and demecolcine are joined in culture medium. Stop adding when cell is cultivated a certain amount of ethidium bromide (EB) can make division mutually in chromosomal length longer, band line is more clear, have save time, labour-saving advantage, and accuracy is higher, has application prospect widely in field of medical examination.
Description
Technical field
The invention belongs to biological technical field, particularly in a kind of field of medical examination for chromosome karyotype analysis marrowThe method of G band preparation.
Background technology
The aobvious band of G is because chromosome is mainly shown band after Giemsa dyeing, therefore be referred to as G banding technique, its instituteThe band line showing is distributed on whole chromosome. People will use various method, and process and dye with different dyestuffsAfter body sample, make to occur on every chromosome light and dark, or the technology of depth different band line is called banding technique (bandingTechnique). Since 1970's, banding technique has obtained very great development, and (Q is with, G in numerous banding techniquesBand, C band, R band, T band), G band is a kind of banding pattern being widely used at present.
Research finds, human chromosome sample, after the agent treatment such as trypsase, Na0H, citrate or urea, then is usedGiemsa dyeing, can make to demonstrate the band that the depth replaces on every chromosome, and Here it is, and chromosomal G is with. Every chromosomeThere is its comparatively constant band line feature, so after the aobvious band of G, can identify comparatively accurately every chromosome, and can find to dyeTrickleer structural aberration on colour solid.
In recent years, along with molecular biology and cytogenetic development, the karyotyping of bone marrow stain body is at hematological systemIn diagnosis, treatment and the prognosis of disease, bring into play more and more important effect. The preparation of bone marrow stain body is a large amount of because having in marrowThe interference of lipochondrion, in marrow the cell cycle of various clones fixing, disunity, is difficult to treat with a certain discrimination and makesBone marrow stain body di is low, and chromosome is short and thick, and decentralization is poor, and cost is higher.
Therefore, set up a kind of division marrow G band system of the feature such as many, good dispersion degree,, moderate length clear with line mutually that hasMake method particularly important.
Summary of the invention
In order to overcome the problem of existence of the prior art, the invention provides a kind of chromosome karyotype analysis marrow G bandPreparation method, comprises step:
(A) inoculation: bone marrow cell is inoculated in bone marrow cell culture medium;
(B) stop cultivating: ethidium bromide and demecolcine are joined in the described culture medium of step (A);
(C) collect bone marrow cell culture, for chromosome sectioning;
(D) chromosome specimen film-making: obtain the chromosome specimen with the aobvious band of G after utilizing dyeing.
Further, the concentration of ethidium bromide is 1.5～5.5mg/ml, and the concentration of demecolcine is 8～15 μ g/ml.
Further, taking culture medium consumption as 5ml, add the ethidium bromide of 50 μ l and the demecolcine of 25 μ l.
Further, taking culture medium consumption as 5ml, ethidium bromide and 25 μ l that to add 50 μ l concentration be 3mg/ml are denseDegree is the demecolcine of 12 μ g/ml.
Further, while stopping cultivating, ethidium bromide and demecolcine are added to culture medium, rock evenly latter 37 DEG C,5.0%CO2 incubator is hatched 1 hour.
Further, the method for collecting bone marrow cell culture comprises:
(1) centrifugal acquisition bone marrow cell;
(2) bone marrow cell in step (1) is carried out to hypotonic processing;
(3) bone marrow cell in step (2) is pre-fixed with fixer;
(4) bone marrow cell in step (3) is fixed with fixer;
(5) bone marrow cell after fixing is made to the suspension that concentration is moderate and be with film-making for G.
Further, the method for chromosome sectioning comprises:
A. the preparation of slide;
B. drip sheet;
C. roasting sheet is aging;
D. prepare digestive juice;
E. prepare dyeing liquor;
Further, described dyestuff is Giemsa.
Further, described Giemsa dyestuff mixes use with the phosphate buffer of pH6.8 according to 1:20.
Further, by bone marrow cell with 1～3 × 106The density of individual/ml is inoculated in bone marrow cell culture medium, puts into37℃，5.0%CO2Incubator is cultivated 24 hours.
The invention has the beneficial effects as follows: in the time stopping cell cultivation, add a certain amount of ethidium bromide (EB) can make dyeingBody length increases, and makes in division mutually chromosomal length longer, and band line is more clear, have save time, labour-saving advantage, and accuratelyProperty is higher, has application prospect widely in field of medical examination.
Brief description of the drawings
Fig. 1 utilizes experimental group 1 gained chromosome specimen microscopy result.
Fig. 2 utilizes experimental group 2 gained chromosome specimen microscopy results.
Fig. 3 utilizes experimental group 3 gained chromosome specimen microscopy results.
Fig. 4 utilizes control group gained chromosome specimen microscopy result.
Detailed description of the invention
Below in conjunction with specific embodiment, further set forth the present invention.
Embodiment 1 prepares bone marrow cell and cultivates stop buffer
Bone marrow cell is cultivated stop buffer and is comprised the ethidium bromide of 1.5～5.5mg/ml and the demecolcine of 8～15 μ g/ml.
The compound method of wherein said ethidium bromide and demecolcine can adopt the method described in the present embodiment, also canAdopt other method preparation of this area.
A prepares ethidium bromide
A. prepare stock solution (concentration is 9mg/ml)
In 100ml distilled water, add 0.9g ethidium bromide, magnetic agitation a few hours dissolve completely to guarantee it, then useAluminium foil wrapping container or be transferred in brown bottle, is stored in room temperature.
B. prepare working solution (concentration is 3mg/ml)
Stock solution is with 1:2(EB:ddH2O) it is the working solution of 3mg/ml that dilution proportion becomes concentration.
B prepares demecolcine
A. prepare stock solution (concentration is 120 μ g/ml)
Take 12mg demecolcine, add 8.5g/LNaCl solution 100mL, until completely dissolved, through 5.516 ×104Pa(81bf/in2) after 15min high pressure steam sterilization, keep in Dark Place in 4 DEG C of refrigerators.
B. prepare working solution (concentration is 12 μ g/ml)
Getting 120 μ g/ml demecolcine solution 1mL adds 8.5g/LNaCl solution 9mL to be the demecolcine of 12 μ g/mL.
Embodiment 2 prepares bone marrow stain body sample
The present embodiment is prepared bone marrow cell chromosome sample as follows. Comprise the following steps:
(A) inoculation: bone marrow cell is inoculated in bone marrow cell culture medium;
(B) stop cultivating: the stop buffer described in embodiment 1 is joined in step (A) culture medium;
(C) collect bone marrow cell culture, for chromosome sectioning;
(D) chromosome specimen film-making: obtain the chromosome specimen with the aobvious band of G after utilizing dyeing.
Taking culture medium consumption as 5ml, add the ethidium bromide that 50 μ l concentration are 1.5～5.5mg/ml in the present embodimentWith 25 μ l concentration be the demecolcine of 8～15 μ g/ml.
While stopping cultivating in the present embodiment, ethidium bromide and demecolcine are added to culture medium, rock evenly latter 37 DEG C,5.0%CO2Incubator is hatched 1 hour.
Wherein by bone marrow cell with 1～3 × 106The density of individual/ml is inoculated in marrow culture medium, puts into 37 DEG C, 5.0%CO2Incubator is cultivated 24 hours. Gained marrow culture can be used for bone marrow stain system sheet.
Wherein, collect bone marrow cell culture and can collect as follows, also can receive by other conventional method of this areaCollection.
(1) the gentle blake bottle that rocks, and culture is proceeded in corresponding 15ml centrifuge tube. Tighten cultivation bottle cap, andGuarantee that sample is not mixed mutually. The centrifugal 10min of 1,000rpm. Suck supernatant, leave approximately 0.5 and mix to 1.0ml vortex.
(2) hypotonic: the 0.075MKCl solution that adds 10ml37 DEG C of incubator preheating. Vortex or repeatedly put upside down and make several times itWith sample blending, 20～30min is hatched in 37 DEG C of water-baths, centrifuge tube is rolled three times during this time so that cell is hypotonic evenly.
(3) pre-fix: after hypotonic end, add the fixer (methyl alcohol: glacial acetic acid=3:1) of 1ml, tighten lid also repeatedlyPut upside down three times. The centrifugal 10min of 1,000rpm. Suck supernatant, leave approximately 0.5 to 1.0ml.
(4) after sediment vortex is mixed, dropwise add the fresh fixer of 8ml.
(5) vortex mixes the rear centrifugal 10min of 1,000rpm. Suck supernatant, leave approximately 0.5 to 1.0ml.
(6) add the fresh fixer of 8ml after mixing cell precipitation.
(7) with 5 and 6.
(8) vortex mixes the rear centrifugal 10min of 1,000rpm. Suck supernatant, stay appropriate fixer, and add several iceAcetic acid to be to make the cell suspension that concentration is suitable, leaves standstill film-making after 15min.
Wherein the chromosome specimen flaking method in the present embodiment is as follows:
A. the preparation of slide: in advance slide is used to 1%HCl soaked overnight, be dipped in 95% after rinsing with a large amount of clear waterFor subsequent use in ethanol. Before using, the slide soaking is taken out, after cleaning with a large amount of clear water, be positioned in 2-8 DEG C of refrigerator stand-by.
B. drip sheet: draw after cell suspension, dropper is placed in to certain height, drip 4-5 and drip cell suspension on slide, makeCell flows to slide mark end distally. Suitably overdo, help Chromosome spread. A general patient is dripped sheet 1-2 and is opened.
C. roasting sheet is aging: be placed in that 60 DEG C of oven for baking are spent the night or 80 DEG C of bakings 1 hour.
D. prepare pancreatin: fresh preparation 50ml0.3% trypsase (with the dilution of HANKS buffer solution) solution is placed in and dyes sheetIn cylinder, in 37 DEG C of water baths more than preheating half an hour. Trypsase and HANKS buffer solution are all purchased from Invitrogen company.
E. prepare Giemsa dye liquor: face the used time Gimesa stoste is mixed and made according to 1:20 with the phosphate buffer of pH6.8With. The a that concrete compound method can follow these steps to and b, also can be by other conventional method preparation of this area.
A. prepare stock solution
Giemsa powder 1g
Pure glycerin 66ml
Methyl alcohol 66ml
First Giemsa powder is placed in to mortar and adds a small amount of glycerine, fully grind, be agranular pasty state, then by whole glycerineAdd, put into 56 DEG C of incubators 2 hours, then add methyl alcohol, be stored in brown bottle; Use after general two weeks as well;
B. preparation work liquid
Facing the used time mixes with the phosphate buffer of pH6.8 the stock solution in a step according to 1:20.
F. with after trypsinization dyeing for chromosome karyotype analysis.
Embodiment 3 contrast experiments
Get the bone marrow cell of same sample after cultivating, carry out contrast experiment. Adopt the method for embodiment 1 and embodiment 2Set up experimental group 1, experimental group 2 and 3, three groups of experimental group of experimental group to adopt respectively the cell of variable concentrations to cultivate stop buffer, wherein:
The stop buffer formula of experimental group 1 is:
Ethidium bromide 1.5mg/ml
Demecolcine 8 μ g/ml
The stop buffer formula of experimental group 2 is:
Ethidium bromide 5.5mg/ml
Demecolcine 15 μ g/ml
The stop buffer formula of experimental group 3 is:
Ethidium bromide 3mg/ml
Demecolcine 12 μ g/ml
Set up control group, wherein:
The stop buffer formula of control group is:
Demecolcine 12 μ g/ml
The step that control group is prepared bone marrow cell chromosome sample comprises: (A) inoculation: bone marrow cell is inoculated into marrow thinIn born of the same parents' culture medium; (B) stop cultivating: stop buffer is joined in step (A) culture medium, taking culture medium consumption as 5ml, addThe demecolcine of 25 μ l; (C) collect bone marrow cell culture, for chromosome sectioning; (D) chromosome specimen film-making: utilize and dyeAfter material dyeing, obtain the chromosome specimen with the aobvious band of G.
In Microscopic observation film-making result. The microscope model using is: LeicaDM2500. Fig. 1 to Fig. 4 is respectively experimentThe microscopy result figure of group 1, experimental group 2, experimental group 3 and control group. From the microscopy photo of Fig. 1 to 3, very clearly see, useThe inventive method is carried out G band colour developing, and in division mutually, chromosomal length is longer, and band line is more clear, thereby when diagnosis, more economizesShi Shengli, diagnostic result is more accurate.
Claims (6)
1. a chromosome karyotype analysis marrow G band preparation method, comprises step:
(A) inoculation: by bone marrow cell with 1～3 × 106The density of individual/ml is inoculated in bone marrow cell culture medium, puts into 37 DEG C,5.0％CO2Incubator is cultivated 24 hours;
(B) stop cultivating: in the time stopping cultivating, taking culture medium consumption as 5ml, be 1.5～5.5mg/ by 50 μ l concentration simultaneouslyThe ethidium bromide of ml and 25 μ l concentration are the demecolcine of 8～15 μ g/ml, join in the described culture medium of step (A), rockEvenly latter 37 DEG C, 5.0%CO2Incubator is hatched 1 hour;
(C) collect bone marrow cell culture, for chromosome sectioning;
(D) chromosome specimen film-making: obtain the chromosome specimen with the aobvious band of G after utilizing dyeing.
2. preparation method according to claim 1, is characterized in that, taking culture medium consumption as 5ml, adds 50 μ l concentrationFor ethidium bromide and the 25 μ l concentration of 3mg/ml are the demecolcine of 12 μ g/ml.
3. preparation method according to claim 1, is characterized in that, the method for collecting bone marrow cell culture comprises:
(1) leniently rock blake bottle, and culture is proceeded in corresponding 15ml centrifuge tube, tighten cultivation bottle cap, and guaranteeSample is not mixed mutually, and the centrifugal 10min of 1,000rpm, sucks supernatant, leaves 0.5 to 1.0ml vortex and mixes;
(2) hypotonic: add the 0.075MKCl solution of 10ml37 DEG C of incubator preheating, vortex or repeatedly put upside down makes itself and sample several timesProduct mix, and 20～30min is hatched in 37 DEG C of water-baths, centrifuge tube are rolled three times during this time so that cell is hypotonic evenly;
(3) pre-fix: after hypotonic end, add the fixer of 1ml methyl alcohol: glacial acetic acid=3:1, tighten lid and repeatedly put upside down threeInferior, the centrifugal 10min of 1,000rpm; Suck supernatant, leave 0.5 to 1.0ml;
(4) after sediment vortex is mixed, dropwise add the fresh fixer of 8ml;
(5) vortex mixes the rear centrifugal 10min of 1,000rpm, sucks supernatant, leaves 0.5 to 1.0ml;
(6) add the fresh fixer of 8ml after mixing cell precipitation;
(7) with step 5 and 6;
(8) vortex mix after the centrifugal 10min of 1,000rpm, suck supernatant, stay appropriate fixer, and add several glacial acetic acid withMake the cell suspension that concentration is suitable, film-making after standing 15min.
4. preparation method according to claim 1, is characterized in that, the method for chromosome sectioning comprises:
A. the preparation of slide: in advance slide is used to 1%HCl soaked overnight, be dipped in 95% second after rinsing with a large amount of clear waterFor subsequent use in alcohol, before using, the slide soaking is taken out, after cleaning with a large amount of clear water, be positioned in 2-8 DEG C of refrigerator stand-by;
B. drip sheet: draw after cell suspension, dropper is placed in to certain height, drip 4-5 and drip cell suspension on slide, make cellFlow to slide mark end distally, suitably overdo, help Chromosome spread;
C. roasting sheet is aging: be placed in that 60 DEG C of oven for baking are spent the night or 80 DEG C of bakings 1 hour;
D. prepare pancreatin: by the dilution of trypsase HANKS buffer solution, fresh preparation 50ml0.3% trypsin solution is placed inDye in sheet cylinder, in 37 DEG C of water baths, more than preheating half an hour, trypsase and HANKS buffer solution are all purchased from Invitrogen public affairsDepartment;
E. prepare Giemsa dye liquor: face the used time Gimesa stoste is mixed to use with the phosphate buffer of pH6.8 according to 1:20;
F. use trypsinization, dyeing.
5. preparation method according to claim 1, is characterized in that, described dyestuff is Giemsa.
6. preparation method according to claim 1, is characterized in that, the compound method of described ethidium bromide comprises:
A. the stock solution that compound concentration is 9mg/ml
In 100ml distilled water, add 0.9g ethidium bromide, magnetic agitation a few hours dissolve completely to guarantee it, then use aluminium foilWrapping container or be transferred in brown bottle, is stored in room temperature;
B. the working solution that compound concentration is 3mg/ml
Stock solution EB:ddH2O becomes the working solution of concentration as 3mg/ml taking the dilution proportion of 1:2;
The compound method of described demecolcine comprises:
A. compound concentration is the stock solution of 120 μ g/ml
Take 12mg demecolcine, add 8.5g/LNaCl solution 100mL, until completely dissolved, through 5.516 × 104Pa15minAfter high pressure steam sterilization, keep in Dark Place in 4 DEG C of refrigerators;
B. compound concentration is the working solution of 12 μ g/ml
Getting 120 μ g/ml demecolcine solution 1mL adds 8.5g/LNaCl solution 9mL to be the demecolcine of 12 μ g/mL.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103983497B (en) * | 2014-06-12 | 2016-04-20 | 周口师范学院 | The preparation method of turbellarian worm chromosome specimen |
| CN104792599A (en) * | 2015-05-11 | 2015-07-22 | 中国医学科学院血液病医院（血液学研究所） | Preparation method of chromosome R banding |
| CN107063788A (en) * | 2017-01-06 | 2017-08-18 | 中国人民解放军第八医院 | A kind of preparation method of lymphocyte chromosome G bands |
| CN108168968A (en) * | 2017-12-14 | 2018-06-15 | 济南金域医学检验中心有限公司 | A kind of production method of marrow chromosome G band |
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