CN108396008A - A kind of abalone haemocyte processing method - Google Patents
A kind of abalone haemocyte processing method Download PDFInfo
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- CN108396008A CN108396008A CN201810075334.3A CN201810075334A CN108396008A CN 108396008 A CN108396008 A CN 108396008A CN 201810075334 A CN201810075334 A CN 201810075334A CN 108396008 A CN108396008 A CN 108396008A
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Abstract
The invention discloses a kind of abalone haemocyte processing methods, and 1ml anticoagulation protecting agents are added first in 2ml centrifuge tubes；Then abalone abdominal foot portion blood sinus is scratched with scalpel, abalone, which is tilted 20 30 °, to be placed on ice so that blood is more easy to flow out；It waits for that blood flows out, is taken out with asepsis injector in the centrifuge tube that 1ml blood is directly injected into after anticoagulation protecting agent is added；With in a kind of blood filter filtering blood for flow cytomery to new 2ml centrifuge tubes；Again through 18 DEG C, 3000rpm centrifuges 10min, removes supernatant；Bao hemolymph suspension finally is made with 1ml bacteriological filtrations seawater again suspension haemocyte.This method can effectively extend the agglutination time of haemocyte by using suitable blood anticoagulant protective agent and blood filter, the impurity in fast filtering blood, improve the purity of blood, reduce experimental error, keep experimental result more accurate.
Description
Technical field
The present invention relates to abalone haemocyte processing technology fields, and in particular to a kind of abalone haemocyte processing method.
Background technology
For abalone due to full of nutrition, economic value is high, becomes the important sea-farming kind in China.Currently, biological real
Field is tested, mode being uniformly processed directed entirely to abalone haemocyte there is no a kind of.The a variety of different modes of people's generally use
To handle the haemocyte of abalone.In the case where being not added with anticoagulation protecting agent, abalone haemocyte is once in vitro, i.e. a large amount of in a few minutes
Agglutination.Even if the abalone haemocyte that certain anticoagulation protecting agents are added also can mortality in a short time.Reality is not required nothing more than in this way
Testing will complete in a short time, also result in experimental result be affected by systematic error, experimental cost increase, experimental result
The problems such as inaccurate.Therefore haemocyte is quickly handled, it is ensured that abalone haemocyte is not aggregated in an experiment very within a certain period of time
It is important.
It is presently used for shellfish (abalone) blood of flow cytomery, there is no complete sets or regular processing to fill
It sets, the blood being collected into generally is carried out flow cytomery by people without filtration treatment with regard to direct.The result shows that not passing through
The blood for crossing processing contains more impurity, and the image showed is that a comparison is chaotic, rambling scatter plot.In addition,
Untreated cell group be easy to cause detector blocking, needs to take a long time to clean (10-40min) detector,
It can continue test sample.Not only delay the process of detection in this way, also invisible is caused testing expense to increase (200 yuan/h).So
It is also very necessary using suitable blood filter in abalone haemocyte processing procedure.
Invention content
The purpose of the present invention is to provide abalone haemocyte processing method, by using suitable blood anticoagulant protective agent and
Blood filter, can effectively extend the agglutination time of haemocyte, and the impurity in fast filtering blood improves the pure of blood
Degree reduces experimental error, keeps experimental result more accurate.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of abalone haemocyte processing method, which is characterized in that the treating method comprises following steps：
(1) 1ml anticoagulation protecting agents are added first in 2ml centrifuge tubes；
(2) and then with scalpel scratch abalone abdominal foot portion blood sinus, by abalone inclination 20-30 ° be placed on ice so as to blood more
Easily outflow；
(3) it waits for that blood flows out, the centrifuge tube that 1ml blood is directly injected into after anticoagulation protecting agent is added is taken out with asepsis injector
In；
(4) it is centrifuged to new 2ml with liquid in a kind of blood filter filtration step (3) for flow cytomery
Guan Zhong；
(5) again through 18 DEG C, 3000rpm centrifuges 10min, removes supernatant；
(6) Bao hemolymph suspension is made in the haemocyte finally to be suspended again with 1ml bacteriological filtrations seawater by step (5) processing.
In above-mentioned abalone haemocyte processing method, the anticoagulation protecting agent consists of the following compositions：Sodium chloride 2.45~
2.55g, EDTA0.9~1.1g, two ethanesulfonic acid 0.22~0.24g and 0.1mM PBS 100ml of Isosorbide-5-Nitrae-piperazine.
Preferably, the anticoagulation protecting agent consists of the following compositions：Sodium chloride 2.5g, EDTA 1g, Isosorbide-5-Nitrae-piperazine diethyl
Sulfonic acid 0.23g and 0.1mM PBS 100ml.
In above-mentioned abalone haemocyte processing method, the preparation method of the anticoagulation protecting agent is：
(1) 0.1mM PBS buffer solution is poured into beaker, then 3-5 points of solid sodium chloride powder stirring is added into beaker
Then clock adds two ethanesulfonic acid powder of Isosorbide-5-Nitrae-piperazine and is sufficiently stirred and dissolve to obtain mixed solution A until sodium chloride all dissolvings；
(2) mixed solution A is put into 38-40 DEG C of water-bath and is heated, be slow added into EDTA, stirred while adding, directly
It is completely dissolved to obtain mixed solution B to EDTA；
(3) mixed solution B is gone to and uses magnetic stirrer 0.5-1h under room temperature state, each component is made fully to be dissolved into
In PBS buffer solution；
(4) superclean bench opens ultraviolet-sterilization function 30-35min, then on superclean bench that mixed solution B is first
It is put into the blue lid bottle of sterilizing with 0.45um sterile filters filtering and impurity removings, then with 0.22um sterile filters by liquid mistake in blue lid bottle
Bacterium is filtered out, abalone blood cell anticoagulation protecting agent is finally obtained.
Preferably, in the preparation method of above-mentioned abalone blood cell anticoagulation protecting agent, water-bath heats in the step (2)
Temperature is 40 DEG C.
Preferably, in the preparation method of above-mentioned abalone blood cell anticoagulation protecting agent, magnetic stirring apparatus in the step (3)
Mixing time is 0.5h.
Above-mentioned abalone blood cell anticoagulation protecting agent can be stored in spare in the blue lid bottle of sterilizing at 4 DEG C or under room temperature.
In addition, a kind of blood filter of blood filter, including fixed station, montant, filter device and article-holding device,
Article-holding device is positioned over the lower section of filter device, and filter device is mounted on by trim ring bar on montant, and trim ring bar is adjusted by height
It saves button to be mounted on montant, montant is mounted on fixed station, and filter device includes upper trim ring and lower clamping ring, upper trim ring and lower clamping ring
Connected with trim ring bar.It is provided with bolting silk between the upper trim ring and lower clamping ring.Fall the impurity in blood using silk cover filtering.
Wherein, there are four clinchings, four clinchings to be distributed on the periphery of the upper trim ring for setting on the upper trim ring.Four pressures
Button can firmly withhold upper trim ring on lower clamping ring, while can clasp the bolting silk between trim ring and lower clamping ring.
Further, bin net is provided on the upper trim ring, there are four square nets for the bin netting gear.Utilize bin
Bolting silk is spatially divided into four parts by net.
Further, the article-holding device includes storage box and centrifuge tube, and the centrifuge tube is placed in storage box.
Preferably, it is provided with cylindrical grooves in the storage box, ice cube is placed in the cylindrical grooves.Use ice cube
Maintain the low temperature environment needed for filtering blood.
In blood filter above-mentioned, there are two spacers, two spacers vertically to hand over for setting in the cylindrical grooves
Fork setting.Cylindrical grooves, are spatially divided into four sulculuses by two spacers being arranged by square crossing, four sulculuses with
Four square nets of bin net correspond, and a centrifuge tube is placed in each sulculus, consequently facilitating label sample, Bu Huizao
At obscuring.
In blood filter above-mentioned, the material of the clinching is plastics.
In blood filter above-mentioned, the material of the upper trim ring and lower clamping ring is stainless steel.
In blood filter above-mentioned, the fixed station is the iron fixed station of diamond shape.The iron fixed station of diamond shape can ensure
The stability of the present invention, will not topple over because positioned at the article-holding device of montant side.
Advantages of the present invention：
By above-mentioned processing method treated abalone haemocyte, since anticoagulation protecting agent is added, haemocyte can after taking out
Do not assemble in a long time agglomerating, still keeps the integrality and independence of cell, and after blood filter filters,
Impurity greatly reduces in blood, so treated that cost can be obtained more in haemocyte in subsequent experimental by this processing method
It is low, the more accurate experimental result of data.
Description of the drawings
Fig. 1 is blood filter structural schematic diagram；
Fig. 2 is article-holding device structural schematic diagram.
Reference numeral：1- fixed stations, 2- montants, 3- filter devices, 4- article-holding devices, 5- trim ring bars, the upper trim rings of 6-, 7-
Lower clamping ring, 8- clinchings, 9- storage boxes, 10- centrifuge tubes, 11- grooves, 12- spacers, 13- bin nets, 14- height-adjusting knobs.
Fig. 3 is that (no anticoagulation protecting agent group is for agglutination situation after the in vitro 1.5h of abalone haemocyte in each anticoagulation protecting agent
Control group)；
Fig. 4 is the agglutination situation in each anticoagulation protecting agent after the in vitro 3h of abalone haemocyte；
Fig. 5 is the agglutination situation in each anticoagulation protecting agent after the in vitro 4h of abalone haemocyte；
Fig. 6 is the agglutination situation in each anticoagulation protecting agent after the in vitro 5h of abalone haemocyte；
In Fig. 3-6, arrow meaning is cell agglutination phenomenon.
Specific implementation mode
The present invention will be further explained by specific embodiment below, it being understood, however, that can be with each
Kind form realizes the present invention without should be limited by embodiments set forth here.It is to be able on the contrary, providing these embodiments
It is best understood from the present invention, and the scope of the present invention can be completely communicated to those skilled in the art.
"comprising" or " comprising " as mentioned in working as in specification in the whole text and claim are an open language, therefore are answered
It is construed to " including but not limited to ".Specification subsequent descriptions are to implement the better embodiment of the present invention, and so description is
For the purpose of the rule of specification, it is not limited to the scope of the present invention.Protection scope of the present invention is when regarding appended power
Profit requires subject to institute's defender.
Embodiment 1
A kind of blood filter, as shown in Figure 1, 2, including fixed station 1, montant 2, filter device 3 and article-holding device 4, institute
The lower section that article-holding device 4 is positioned over filter device 3 is stated, the filter device 3 is mounted on by trim ring bar 5 on montant 2, trim ring bar
5 are mounted on by height-adjusting knob 14 on montant 2, and montant 2 is mounted on fixed station 1, and the filter device 3 includes upper trim ring 6
With lower clamping ring 7, the upper trim ring 6 and lower clamping ring 7 are connected with montant 2.It is provided with bolting silk between upper trim ring 6 and lower clamping ring 7.Lattice
Mouth net 13 is filtered blood by bolting silk.
Further, it for the ease of replacing the bolting silk being arranged between upper trim ring 6 and lower clamping ring 7, is provided on upper trim ring 6
Four clinchings 8, four clinchings 8 are distributed on the periphery of the upper trim ring 6.Four clinchings 8 can firmly withhold upper trim ring 6
On lower clamping ring 7, while the bolting silk between trim ring 6 and lower clamping ring 7 can be clasped.
Further, bin net 13 is provided on upper trim ring 6, there are four square nets for the tool of bin net 13.Utilize bin net
Bolting silk is divided into four parts by 13.
Specifically, article-holding device 4 includes storage box 9 and centrifuge tube 10, centrifuge tube 10 is placed in storage box 9.Centrifuge tube
10 for containing filtered blood.
Further, it is provided with cylindrical grooves 11 in storage box 9, ice cube is placed in the cylindrical grooves 11.With
Ice cube maintains the low temperature environment needed for filtered blood.
Further, there are two spacer 12, two 12 square crossing of spacer settings for setting in cylindrical grooves 11.
Cylindrical grooves 11 are split into four sulculuses, four sulculuses and bin net by two spacers 12 being arranged by square crossing
13 four square nets correspond, and a centrifuge tube 10 is placed in each sulculus, consequently facilitating label sample, will not cause
Obscure.
Specifically, the material of clinching 8 is plastics.The material of upper trim ring 6 and lower clamping ring 7 is stainless steel.Fixed station 1 is water chestnut
The iron fixed station of shape.The iron fixed station of diamond shape can ensure the stability of the utility model, will not be because of setting positioned at 2 side of montant
Object device 4 and topple over.
When bolting silk is placed between upper trim ring 6 and lower clamping ring 7, bolting silk is divided by bin net 13 for four parts, with removing needle
The syringe or liquid-transfering gun draw blood of head, adjust height-adjusting knob 14, it are made just to be directed at storage box 9 (wherein putting ice)
Four centrifuge tubes 10, alignment a portion bolting silk are injected down, liquid by bolting silk enter below face in storage box 9 from
Heart pipe 10；Each section bolting silk, when the bolting silk being finished in circle, is opened clinching 8, changes a brand-new bolting silk with once.Pass through
The blood of this blood filter filtering, can filter out the impurity in blood, improve the purity of blood, keep it thin through overflow-type
After the detection of born of the same parents' instrument, regular, clustering cell scatter plot can be obtained.
Embodiment 2
A kind of abalone haemocyte processing method, the treating method comprises following steps：
(1) 1ml anticoagulation protecting agents are added first in 2ml centrifuge tubes；
(2) and then with scalpel scratch abalone abdominal foot portion blood sinus, by abalone inclination 20-30 ° be placed on ice so as to blood more
Easily outflow；
(3) it waits for that blood flows out, the centrifuge tube that 1ml blood is directly injected into after anticoagulation protecting agent is added is taken out with asepsis injector
In；
(4) it uses in the liquid to new 2ml centrifuge tubes in the blood filter filtration step (3) in embodiment 1；
(5) again through 18 DEG C, 3000rpm centrifuges 10min, removes supernatant；
(6) Bao hemolymph suspension is made in the haemocyte finally to be suspended again with 1ml bacteriological filtrations seawater by step (5) processing.
Wherein, the anticoagulation protecting agent consists of the following compositions：Sodium chloride 2.5g, EDTA 1g, Isosorbide-5-Nitrae-piperazine diethyl sulphur
Sour 0.23g and 0.1mM PBS 100ml.
The preparation method of above-mentioned abalone blood cell anticoagulation protecting agent is specially：
(1) it takes 100ml 0.1mM PBS buffer solution to pour into beaker, then 2.5g solid chlorine sodium powders is added into beaker
End stirring 3 minutes, until sodium chloride all dissolve, then add two ethanesulfonic acid powder of 0.23g Isosorbide-5-Nitraes-piperazine be sufficiently stirred it is molten
Solve mixed solution A；
(2) and then mixed solution A is put into 40 DEG C of water-baths and is heated, be slow added into EDTA 1g, stirred when being added
It mixes, until EDTA is completely dissolved to obtain mixed solution B；
(3) mixed solution B is gone to and uses magnetic stirrer 0.5h under room temperature state, each component is made fully to be dissolved into
In PBS buffer solution；
(4) superclean bench opens ultraviolet-sterilization function 30min, then first uses mixed solution B on superclean bench
0.45um sterile filters filtering and impurity removings are put into the blue lid bottle of sterilizing, then are filtered liquid in blue lid bottle with 0.22um sterile filters
Degerming, the abalone blood cell anticoagulation protecting agent then obtained.
Embodiment 3
A kind of abalone haemocyte processing method, the treating method comprises following steps：
(1) 1ml anticoagulation protecting agents are added first in 2ml centrifuge tubes；
(2) and then with scalpel scratch abalone abdominal foot portion blood sinus, by abalone inclination 20-30 ° be placed on ice so as to blood more
Easily outflow；
(3) it waits for that blood flows out, the centrifuge tube that 1ml blood is directly injected into after anticoagulation protecting agent is added is taken out with asepsis injector
In；
(4) it uses in the liquid to new 2ml centrifuge tubes in the blood filter filtration step (3) in embodiment 1；
(5) again through 18 DEG C, 3000rpm centrifuges 10min, removes supernatant；
(6) Bao hemolymph suspension is made in the haemocyte finally to be suspended again with 1ml bacteriological filtrations seawater by step (5) processing.
The anticoagulation protecting agent, consists of the following compositions：Sodium chloride 2.55g, EDTA 1.1g, two ethanesulfonic acid of Isosorbide-5-Nitrae-piperazine
0.24g and 0.1mM PBS 100ml.
The preparation method of above-mentioned abalone blood cell anticoagulation protecting agent is specially：
(1) it takes 100ml 0.1mM PBS buffer solution to pour into beaker, then 2.55g solid chlorine sodium powders is added into beaker
End stirring 4 minutes, until sodium chloride all dissolves, then, addition 0.24g Isosorbide-5-Nitraes-two ethanesulfonic acid powder of piperazine is sufficiently stirred molten
Solve mixed solution A；
(2) and then mixed solution A is put into 39 DEG C of water-baths and is heated, be slow added into 1.1g EDTA, stirred when being added
It mixes, until EDTA is completely dissolved to obtain mixed solution B；
(3) mixed solution B is gone to and uses magnetic stirrer 45min under room temperature state, each component is made fully to be dissolved into
In PBS buffer solution；
(4) superclean bench opens ultraviolet-sterilization function 32min, then first uses mixed solution B on superclean bench
0.45um sterile filters filtering and impurity removings are put into the blue lid bottle of sterilizing, then are filtered liquid in blue lid bottle with 0.22um sterile filters
Degerming, the abalone blood cell anticoagulation protecting agent then obtained.
Embodiment 4
A kind of abalone haemocyte processing method, the treating method comprises following steps：
(1) 1ml anticoagulation protecting agents are added first in 2ml centrifuge tubes；
(2) and then with scalpel scratch abalone abdominal foot portion blood sinus, by abalone inclination 20-30 ° be placed on ice so as to blood more
Easily outflow；
(3) it waits for that blood flows out, the centrifuge tube that 1ml blood is directly injected into after anticoagulation protecting agent is added is taken out with asepsis injector
In；
(4) it uses in the liquid to new 2ml centrifuge tubes in the blood filter filtration step (3) in embodiment 1；
(5) again through 18 DEG C, 3000rpm centrifuges 10min, removes supernatant；
(6) Bao hemolymph suspension is made in the haemocyte finally to be suspended again with 1ml bacteriological filtrations seawater by step (5) processing.
The anticoagulation protecting agent, consists of the following compositions：Sodium chloride 2.45g, EDTA0.9g, two ethanesulfonic acid of Isosorbide-5-Nitrae-piperazine
0.22g and 0.1mM PBS 100ml.
The preparation method of above-mentioned abalone blood cell anticoagulation protecting agent is specially：
(1) it takes 100ml 0.1mM PBS buffer solution to pour into beaker, then 2.45g solid chlorine sodium powders is added into beaker
End stirring 5 minutes, until sodium chloride all dissolves, then, addition 0.22g Isosorbide-5-Nitraes-two ethanesulfonic acid powder of piperazine is sufficiently stirred molten
Solve mixed solution A；
(2) mixed solution A is put into 38 DEG C of water-baths and is heated, be slow added into 0.9g EDTA, stir while adding,
Until EDTA is completely dissolved to obtain mixed solution B；
(3) mixed solution B is gone to and uses magnetic stirrer 1h under room temperature state, each component is made fully to be dissolved into PBS
In buffer solution；
(4) superclean bench opens ultraviolet-sterilization function 35min, then first uses mixed solution B on superclean bench
0.45um sterile filters filtering and impurity removings are put into the blue lid bottle of sterilizing, then are filtered liquid in blue lid bottle with 0.22um sterile filters
Degerming, the abalone blood cell anticoagulation protecting agent then obtained.
In order to further prove that beneficial effects of the present invention, applicant have also carried out following experiment：
One, different anticoagulation protecting agent anticoagulating active comparisons
No. 1 anticoagulation protecting agent formula (anticoagulation protecting agent formula of the invention, be denoted as AP)：Sodium chloride 2.5g, 0.1mM PBS
100ml, EDTA 1g, two ethanesulfonic acid 0.23g of Isosorbide-5-Nitrae-piperazine；
No. 2 anticoagulation protecting agent formulas：Sodium chloride 2.5g, 0.1mM PBS 100ml, EDTA 1g；
No. 3 anticoagulation protecting agent formulas：Sodium chloride 2.5g, 0.1mM PBS 100ml, EDTA 1g, sodium oxalate 0.15g.
Abalone haemocyte is pressed 1 from different anticoagulation protecting agents (1,2, No. 3 anticoagulation protecting agent) first：1 volume mixture, room
The agglutination situation of haemocyte is shown in Table 1 and Fig. 3 after temperature holding 1.5h, and the pure Bao blood for not adding anticoagulation protecting agent is increased in this comparison
Cell is as a contrast.
Agglutination situation of the 1 abalone haemocyte of table in different anticoagulation protecting agents
Note：Agglutination group number refers to average each visual field cell mass number in 20 visuals field of random observation.
Microscopically observation, the abalone haemocyte for being not added with anticoagulation protecting agent are aggregated, and multiple cell agglutination groups occur, and
The cell agglutination number of each agglutination group is more than 20 cells.Abalone haemocyte is presented in AP and is uniformly distributed, in random observation
Haemocyte group number is less than 2 in 20 visuals field, and the blood cell count of each cell mass is less than 3, and anti-freezing collection effect is best.No. 2
The effect of anticoagulation protecting agent is taken second place, and hemagglutination phenomenon generally occurs in No. 3 anticoagulation protecting agents.
Continue on the basis of above-mentioned experiment to add 1,2, No. 3 anticoagulation protecting agent abalone haemocyte anti-freezing situation into
Row observation.At ambient temperature, respectively to keep 3h, 4h, 5h after haemocyte No. 1, No. 2, No. 3 anticoagulation protecting agent agglutination
Situation is compared, and specific comparison result is shown in Table 2 and Fig. 4-6.
Condensation situation of the 2 different time sections abalone haemocyte of table in different anticoagulation protecting agents
From fig. 4, it can be seen that haemocyte is still evenly distributed in AP after 3 hours, haemocyte has becoming for agglutination in the visual field
The cell number of gesture, agglutination only has 3-4.Haemocyte is unevenly distributed in No. 2 anticoagulation protecting agents, and haemocyte has bright in the visual field
The phenomenon that aobvious agglutination, the cell number of agglutination is more than 5, and cotton-shaped impurity phenomenon occurs.Haemocyte is in No. 3 anticoagulation protecting agents
It is unevenly distributed, haemocyte has the phenomenon that apparent agglutination in the visual field, and the cell number of agglutination is more than 10.
From fig. 5, it can be seen that haemocyte distribution uniform in AP after 4 hours, there is a small amount of cell agglutination group to go out in the visual field
It is existing, but the cell number being aggregated is no more than 4, and agglutination phenomenon is not serious.Haemocyte is unevenly distributed in No. 2 anticoagulation protecting agents
It is even, there is apparent cell agglutination group in the visual field, and the cell number being aggregated is more than 8.Haemocyte divides in No. 3 anticoagulation protecting agents
Cloth is extremely uneven, and cell agglutination group is more in the visual field, and the cell number being aggregated is more than 20.
From fig. 6, it can be seen that haemocyte is distributed more uneven in AP after 5 hours, there is a small amount of cell agglutination group in the visual field
The cell number for occurring, but being aggregated is no more than 6, and agglutination phenomenon is not serious.As seen from the figure, haemocyte resists at No. 2 after 5 hours
It is unevenly distributed in solidifying protective agent, there is a large amount of cell agglutination group to occur in the visual field, agglutination phenomenon is more serious.Blood is thin after 5 hours
Born of the same parents' distributed pole in No. 3 anticoagulation protecting agents is uneven, the cell agglutination group more (＞ 22 of cell number that are more, and being aggregated in the visual field
It is a), agglutination phenomenon is serious.
Test proves that at different time sections (0-5h), AP anticoagulation protecting agents are either maintaining cellular morphology, still
It prevents from being all substantially better than anticoagulation protecting agent 2 and 3 in terms of cell agglutination, is to study selection ideal in abalone experiment.
Two, different anticoagulation protecting agent haemocyte death rate comparisons
The measurement of the haemocyte death rate is carried out using flow cytometer.Concrete operation step is as follows：Take out 500ul abalone blood
In (after adding different anticoagulation protecting agents) to new 2ml sterile centrifugation tubes, each addition PI dye liquor 10ul (final concentration 20ug/m1), 20 DEG C
10min is incubated in darkroom.20 DEG C, 400g centrifuges 5min to remove undyed PI reagents.It is resuspended with 600ul sterilizing seawater thin
Born of the same parents are put into 5ml streaming pipes.With Flow cytometry 30 seconds.There is fluorescence signal under the conditions of 630nm, use flow cytometer
The one-parameter histogram of FL2 channels distinguishes dead cell and living cells.
The cell mortality that abalone blood cell measures after being mixed in equal volume with various anticoagulation protecting agents the results are shown in Table 3.
The death rate of 3 haemocyte of table in each anticoagulation protecting agent
From table 3 it is observed that the haemocyte in AP shows the lower death rate in 1.5-5h, and No. 2 and No. 3
The death rate of anti-coagulants is higher than AP.Take the anticoagulant effect of table 1 and 2 into consideration, AP anticoagulation protecting agent effects of the invention are more preferable.
Three, the treatment effect of blood filter of the present invention is utilized
The effect for filtering front and back is compared, after filtering hemolymph with blood filter of the present invention, is eliminated a large amount of miscellaneous
Matter, flow cytometer scavenging period shorten, and analytical error reduces, and obtained experimental result is more accurate.
And machine scavenging period on its abalone blood cell samples and expense are compared, specific comparison result is shown in Table 4.
Machine scavenging period and expense on 4 abalone blood cell samples of table
In order to detect the effect of cell processing, applicant has continued following detection, specific as follows：Using fluidic cell
Instrument this highly sophisticated device surveys the phagocytic activity of haemocyte.It is as follows：Take 500ul Bao hemolymph suspensions to 2ml from
In heart pipe, 88ul fluorescent microspheres liquid (the fluorescent microsphere liquid of fluorescent microsphere 10ul+ filtering sea 490ul → 2%) is added, mixing is equal
Even be placed in 20 DEG C of darkrooms is incubated 60 minutes.The cold anticoagulation protecting agents of 300ul are added into each centrifuge tube and terminate reaction.20
DEG C, 400g centrifuges 5min (fluorescent microsphere not swallowed has been arrived in supernatant), abandons supernatant.600ul bacteriological filtration seawater weights are used again
Sample is poured into 5ml flow cytometers and is measured by new suspension cell, and specific testing result is shown in Table 5.
Influence of 5 cell of the table processing to cell phagocytic activity
As can be seen from Table 5, cell is substantially less than without the phagocytic activity value of anticoagulation protecting agent and filter process
Processing group.This shows that untreated cell can significantly affect phagocytic activity, and finally influences the accuracy of measuring.
And treated cell does not influence the phagocytic activity of cell, and experiment is contributed to obtain accurate measured value.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention belong to the scope of protection of present invention.
Claims (10)
1. a kind of abalone haemocyte processing method, which is characterized in that the treating method comprises following steps：
(1) 1ml anticoagulation protecting agents are added first in 2ml centrifuge tubes；
(2) and then with scalpel abalone abdominal foot portion blood sinus is scratched, abalone inclination is placed on ice so that blood is more easy to flow for 20-30 °
Go out；
(3) it waits for that blood flows out, is taken out with asepsis injector in the centrifuge tube that 1ml blood is directly injected into after anticoagulation protecting agent is added；
(4) with a kind of liquid in blood filter filtration step (3) for flow cytomery to new 2ml centrifuge tubes
In；
(5) again through 18 DEG C, 3000rpm centrifuges 10min, removes supernatant；
(6) Bao hemolymph suspension is made in the haemocyte finally to be suspended again with 1ml bacteriological filtrations seawater by step (5) processing.
2. abalone haemocyte processing method according to claim 1, which is characterized in that the anticoagulation protecting agent by below at
It is grouped as：2.45~2.55g of sodium chloride, 0.9~1.1g of EDTA, Isosorbide-5-Nitrae-piperazine two ethanesulfonic acid 0.22~0.24g and 0.1mM
PBS 100ml。
3. abalone haemocyte processing method according to claim 2, which is characterized in that the preparation side of the anticoagulation protecting agent
Method is：
(1) 0.1mM PBS buffer solution is poured into beaker, then solid sodium chloride powder stirring 3-5 minutes is added into beaker, directly
It is all dissolved to sodium chloride, then adds two ethanesulfonic acid powder of Isosorbide-5-Nitrae-piperazine and be sufficiently stirred and dissolve to obtain mixed solution A；
(2) mixed solution A is put into 38-40 DEG C of water-bath and is heated, be slow added into EDTA, stirred while adding, until
EDTA is completely dissolved to obtain mixed solution B；
(3) mixed solution B is gone to and uses magnetic stirrer 0.5-1h under room temperature state, each component is made fully to be dissolved into PBS
In buffer solution；
(4) superclean bench opens ultraviolet-sterilization function 30-35min, then first uses mixed solution B on superclean bench
0.45um sterile filters filtering and impurity removings are put into the blue lid bottle of sterilizing, then are crossed liquid in blue lid bottle with 0.22um sterile filters and filtered out
Bacterium finally obtains abalone blood cell anticoagulation protecting agent.
4. abalone haemocyte processing method according to claim 1, which is characterized in that the blood filter includes fixing
Platform (1), montant (2), filter device (3) and article-holding device (4), the article-holding device (4) are positioned under filter device (3)
Side, the filter device (3) are mounted on by trim ring bar (5) on montant (2), and the trim ring bar (5) passes through height-adjusting knob
(14) it is mounted on montant (2), the montant (2) is mounted on fixed station (1), and the filter device (3) includes upper trim ring (6)
With lower clamping ring (7), the upper trim ring (6) and lower clamping ring (7) are connected with trim ring bar (5), the upper trim ring (6) and lower clamping ring
(7) bolting silk is provided between.
5. abalone haemocyte processing method according to claim 4, which is characterized in that be provided on the upper trim ring (6)
Four clinchings (8), four clinchings are distributed on the periphery of the upper trim ring (6).
6. abalone haemocyte processing method according to claim 5, which is characterized in that be provided on the upper trim ring (6)
Bin net (13), there are four square nets for bin net (13) tool.
7. abalone haemocyte processing method according to claim 4, which is characterized in that the article-holding device (4) includes setting
Object box (9) and centrifuge tube (10), the centrifuge tube (10) are placed in storage box (9).
8. abalone haemocyte processing method according to claim 7, which is characterized in that be provided in the storage box (9)
Cylindrical grooves (11) are placed with ice cube in the cylindrical grooves (11).
9. abalone haemocyte processing method according to claim 8, which is characterized in that set in the cylindrical grooves (11)
It sets there are two spacer (12), two spacer (12) square crossings are arranged.
10. the material of abalone haemocyte processing method according to claim 1, the clinching (8) is plastics, the upper pressure
The material for enclosing (6) and lower clamping ring (7) is stainless steel, and the fixed station (1) is the iron fixed station of diamond shape.
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| CN113502313A (en) * | 2021-06-25 | 2021-10-15 | 集美大学 | Preparation method of glycopeptide |
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