CN110333357A - A kind of method for drafting of acute hepatic failure mouse liver panimmunity cell characteristic map - Google Patents
A kind of method for drafting of acute hepatic failure mouse liver panimmunity cell characteristic map Download PDFInfo
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Abstract
The present invention relates to the researchs of liver panimmunity cell characteristic map, it is desirable to provide a kind of method for drafting of acute hepatic failure mouse liver panimmunity cell characteristic map.Comprising steps of the extraction of mouse liver panimmunity cell；The label of mass spectrum streaming antibody；The dyeing of immunocyte antibody；Mass spectrum flow cytometer showed.The present invention can remove as far as possible liver parenchymal cell, isolate the mouse liver panimmunity cell of high yield pulp1 on the basis of guaranteeing cell purity.The mouse liver panimmunity cell yield that the present invention separates is being higher than polishing yield, and Cell viability is greater than traditional polishing.The present invention is according to immunocyte feature in liver, lungs, innovatively it is connect with stable metal isotope with antibody using kit, mass spectrum streaming channel is designed based on passage interference minimum principle simultaneously, realize that the classification of liver panimmunity cell and function is described in comprehensive mouse, 43 markers can be at most detected simultaneously, be able to observe that the dynamic of mouse liver panimmunity cell changes.
Description
Invention field
The present invention relates to the researchs of liver panimmunity cell characteristic map, and in particular to a kind of acute hepatic failure mouse liver is complete
The method for drafting of immunocyte characteristic spectrum.
Background technique
Acute hepatic failure is a large amount of necrosiss of liver cells and serious hepatic disorder to occur in the short time, and cause hepatic encephalopathy
One group of bad clinical syndrome.This disease case fatality rate is high, and liver transfer operation is current definite effective treatment means.However, organ is short
It lacks and the factors such as post-transplantation complication greatly limits liver transfer operation in clinical application.In recent years immune formulation, stem cell transplantation etc.
It is quickly grown for the treatment means of pathogenesis, is expected to become new treatment means.
Liver immunity cell largely infiltrates when hepatic failure, such as bone-marrow-derived lymphocyte, T lymphocyte etc., while immunocyte is secreted
A large amount of proinflammatory factor, is further exacerbated by liver failure.Under hepatic failure pathological state, liver panimmunity cell category, ratio are all
Which variation has occurred, these variations are of great significance for development of new immunoregulation medicament.Because of current technical method
Limitation, liver inherent immunity cell and reactive immunocyte characteristic variation are unclear when hepatic failure.Traditional separation side
Method is to mark Liver immunity cell streaming antibody, is separated with conventional flow.Because conventional flow there are sense channel by
Technical restrictions, the experiment greater than 12 colors such as limit and the overlapping of fluorophor emission spectrum can not just carry out, cause thin to Liver immunity
The coverage of born of the same parents' detection is inadequate, and Liver immunity cell subsets characteristic changes when cannot precisely reflect hepatic failure.
Summary of the invention
The technical problem to be solved by the present invention is to overcome deficiency in the prior art, provide a kind of acute hepatic failure mouse
The method for drafting of liver panimmunity cell characteristic map.
In order to solve the technical problem, solution of the invention is:
A kind of method for drafting of acute hepatic failure mouse liver panimmunity cell characteristic map is provided, comprising the following steps:
(1) extraction of mouse liver panimmunity cell
The fresh mouse corpse for taking natural death due to acute hepatic failure, wins liver and perfusion goes out its internal blood；
Half an hour is digested with enzyme mixation after liver is shredded, then carries out density gradient centrifugation and erythrocyte splitting, is obtained pure
Mouse panimmunity cell；
(2) label of mass spectrum streaming antibody
Using the MaxPAR X8 antibody coupling kit of Fluidigm company, the U.S., with stable metal isotope with it is small
The connection of mouse immunocyte surface marker antibody, obtains labelled antibody；
(3) immunocyte antibody dyes
Isolated mouse liver panimmunity cell is incubated for labelled antibody, labelled immune cell；
(4) mass spectrum flow cytometer showed
By the mouse liver panimmunity cell after label, machine is analyzed in mass spectrum streaming, using t-SNE and X-shift algorithm
The data obtained is analyzed；Then by the expression and distribution of multiple detection antibody of different cell subsets in same thermal map
On, and show the expression of unlike signal object and the distribution of different cell subsets by viSNE chart, it is complete that mouse liver is showed with this
The classification map of immunocyte.
In the present invention, the step (1) is specifically included:
(1.1) mouse corpse is wiped with 75% alcohol swab, abdominal cut isolates mouse liver, retains gall-bladder and at least 1
The main blood vessel of centimetre length；
(1.2) with phosphate buffer (phosphate buffer saline, PBS) the portal vein continuous perfusion, punching
Liver is washed, removing internal blood makes liver become white from blood red；
(1.3) gall-bladder and main blood vessel are removed, liver is placed in the culture dish containing phosphate buffer and is embathed；
(1.4) liver is shredded and is placed on containing 4.7 milliliters of improvement Du Shi Eagle's medium (dulbecco ' s
Modified eagle medium, DMEM) and the dissociation pipe of 0.3 milliliter of enzyme mixation in；
(1.5) dissociation pipe is put into German MACS company GentleMACSTMDissociator is dissociated in machine, with m_
Liver_03 mode operation is twice；
(1.6) dissociation pipe is taken out, is placed on the shaking table of 37 DEG C of constant temperature, 220 revs/min of revolving speeds and digests 30 minutes；
(1.7) after digesting, dissociation pipe is again placed in dissociation machine, twice with m_liver_04 mode operation；
(1.8) will dissociation manage in suspension in 100 zut filters to 50 milliliters of centrifuge tubes, with 5 milliliters of phosphate
Buffer is resuspended in dissociation pipe after residue, again filters liquid portion to centrifuge tube；
(1.9) at room temperature, with relative centrifugal force 300g centrifugal treating 10 minutes；
(1.10) after discarding supernatant, 3 milliliters of 36%Percoll cell separating liquids are added into precipitating；At room temperature,
With relative centrifugal force 600g centrifugal treating 15 minutes；
(1.11) supernatant comprising liver cell fragment is discarded, and 2 milliliters of erythrocyte cracked liquid (ACK are added into precipitating
Lysis buffer) 3 minutes are cracked to completely remove red blood cell；Then the termination of addition 5ml phosphate buffer is split red, at 4 DEG C
Under the conditions of with relative centrifugal force 400g centrifugal treating 5 minutes；Supernatant is abandoned, pure mouse liver panimmunity cell is obtained.
In the present invention, in the phosphate buffer include sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate and potassium chloride,
Total concentration is 0.01 mol/L, pH value (pH value) 7.4.
In the present invention, the configuration method of the enzyme mixation are as follows: 12 milligrams of glue are added in 100 milliliters of phosphate buffers
IV, 30 milligram of pronase of protoenzyme and 5 milligrams of deoxyribonuclease Ⅰ powder mix.
In the present invention, before being placed in the liver after shredding in dissociation pipe, it will first contain improvement Du Shi Eagle's medium
It is placed in 37 DEG C of water-baths and preheats 5 minutes with the dissociation pipe of enzyme mixation.
In the present invention, the configuration method of the Percoll cell separating liquid are as follows: first by 1 milliliter of 10 times of phosphate buffer
It is uniformly mixed with 9 milliliters of percoll original solutions, adds 15 milliliters 1 times of phosphate buffer and obtain 25 milliliters of mass concentrations
For 36% Percoll separating liquid.
It include sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate and chlorine in 10 times of phosphate buffers in the present invention
Change potassium, total concentration is 0.1 mol/L, pH value (pH value) 7.4.
In the present invention, in the step (1.9), (1.10) before starting centrifuge, first centrifuge raising speed reduction of speed should be adjusted
It is whole to arrive deep low gear.
In the present invention, the step (2) is specifically included: first being used metal marker substance markers polymer, is obtained and chelate specific gold
Then the polymer of category is used for labelled antibody, obtain labelled antibody.
In the present invention, in the step (2), the stable metal isotope includes: yttrium (Y-89), indium (In-113, In-
115), lanthanum (La-139), praseodymium (Pr-141), neodymium (Nd-142, Nd-143, Nd-144, Nd-145, Nd-146, Nd-148, Nd-
150), samarium (Sm-147, Sm-149, Sm-152, Sm-154), europium (Eu-151, Eu-153), gadolinium (Gd-155, Gd-156, Gd-
157, Gd-158, Gd-160, Gd-197), terbium (Tb-159), dysprosium (Dy-161, Dy-162, Dy-163, Dy-164), holmium (Ho-
165), erbium (Er-166, Er-167, Er-168, Er-170), thulium (Tm-169), ytterbium (Yb-171, Yb-172, Yb-173, Yb-
174, Yb-176), lutetium (Lu-175), platinum (Pt-198), bismuth (Bi-209)；
The antibody has 43, comprising: anti-CD45, anti-CD44, anti-CD19, anti-KI67, anti-
CD24、 anti-MHC II、anti-B220、anti-CD5、anti-CD43、anti-CD38、anti-Ly6G、anti-Ly6C、
anti-CX3CR1、anti-IgD、anti-CD62L、anti-CD11c、anti-TCRd、anti-CD49a、anti-CD80、
anti-BST2、anti-CD25、anti-CD3、anti-F4/80、anti-CD115、anti-iNOS、anti-CXCR3、
anti-CD27、anti-CD103、anti-ICOS、anti-Argnase I、anti-CD49b、anti-Foxp3、anti-
CD127、 anti-CD21、anti-CD23、anti-CD138、anti-CD172a、anti-CTLA-4、anti-SiglecF、
anti-IgM、 anti-CD4、anti-CD8a、anti-CD11b。
Compared with prior art, the beneficial effects of the present invention are:
1, traditional separation method grinds liver organization, according to cell density using being centrifuged, removes hepatic parenchymal cells, this
The isolated immunocyte yield of sample is lower, and can only separate the cell subsets of specific density, cannot efficiently separate liver
Interior all immunocytes.
The present invention can remove as far as possible liver parenchymal cell on the basis of guaranteeing cell purity, isolate high obtain
The mouse liver panimmunity cell of rate.
2, propidium iodide and CD45- fluorescein isothiocynate method validation whether are combined through Flow Cytometry Assay cell,
Propidium iodide positive is dead cell, and the CD45 positive is immunocyte.Compared with the result that polishing in the prior art separates, this hair
Bright isolated mouse liver panimmunity cell yield is being greater than 5 × 106/ only, higher than 1~2.5 × the 10 of polishing yield6/ only,
And Cell viability is greater than 95%, is greater than traditional polishing Cell viability about 85%.
3, MaxPAR X8 antibody coupling kit is commercially produced product, wherein only containing metal isotope and coupling reagent.
The present invention is according to immunocyte feature in liver, lungs, innovatively using the kit with stable metal isotope and business
The connection of antibody purification product, while mass spectrum streaming channel is designed based on passage interference minimum principle, realize comprehensive mouse to liver
The classification of panimmunity cell and function are described.
4, conventional flow detection technique can not carry out the experiment for being greater than 12 colors, cause to cover Liver immunity cell detection
Cover degree is inadequate, and Liver immunity cell subsets characteristic changes when cannot precisely reflect hepatic failure.The present invention can be to mouse liver
Immunocyte carries out systematic detection and classification, can at most detect 43 markers simultaneously, including lineagespecific surface resists
Body, cytokine antibodies and functional antibodies, such as costimulatory molecules antibody.
5, the present invention is able to observe that the dynamic of mouse liver panimmunity cell changes.
Detailed description of the invention
Fig. 1 is the micro- sem observation figure of Liver immunity cellular morphology (20 times of object lens observations) being separated to；
Fig. 2 is hepatic parenchymal cells Wright's staining figure；
Fig. 3 is the Liver immunity cell Wright's staining figure being separated to；
Fig. 4 is the signal of hepatic failure mouse liver panimmunity cell classification map；
Fig. 5 is that the marker expression situation (heatmap) of each group's immunocyte of hepatic failure mouse liver is illustrated.
Specific embodiment
The source of mouse liver used in the present invention is illustrated first:
Mouse liver used in the present invention is taken from the mouse of natural death due to acute hepatic failure of laboratory abandoned, plucks
Take liver should be away from 1 hour death time.The C57BL/6J young rat corpse of 6-8 week old therein, weight 18-25g is selected, is used
After 100 milliliter of 70% ethyl alcohol cleaning, liver is taken out in Biohazard Safety Equipment for follow-up test.In implementation process of the invention, no
In the presence of the operation for killing survival mice, also there is no take mouse living body the disposition of traumatic or Interventional such as split, cut off.
It is sub combined with specific embodiments below, technical solution of the present invention is described in detail.
Instrument and reagent
Dissociation pipe (MACS, German) dissociates machine (MACS, German), constant-temperature table (Thermo, America),
Centrifuge tube (Corning, America), centrifuge (Eppendorf, German), 10 cm dishes (Greiner,
German), 100 micron screens (Corning, America), water-bath (Thermo, America), inverted microscope (Nikon,
Japan), digital slices scanning means (HAMAMATSU, Japan), glass slide (generation is safe, China), mass spectrum flow cytometer
(Fluidigm, America), water purification machine (Thermo, America), the filter (merck of 50 kilodaltons (kDa)
Millipore, America), the filter (merck millipore, America) of 3 kilodaltons (kDa)
DMEM (Gibco, America), PBS (Ji Nuo, China), 10 times of PBS (Gibco, America), clostridiopetidase A IV
(Invitrogen, America), pronase (Roche, America), deoxyribonuclease Ⅰ (Sigma,
America), percoll cell separating liquid (GE Healthcare, Sweden), erythrocyte cracked liquid (Gibco,
America), Rui Shi Ji's nurse Sa dyeing liquor (Bei Suo, China), polymer attachment (Fluidigm, America), the same position of metal
Plain (Fluidigm, America), MaxPAR X8 antibody coupling kit (Fluidigm, America), bovine serum albumin(BSA)
(Suo Laibao, China), sodium azide (Sigma, America), TCEP reducing agent (Thermo, America), antibody confining liquid
(Equitech-Bio, America), fixer (Fluidigm, America), perm buffer (Fluidigm,
America), 20%EQ beads (Fluidigm, America).
(1) separation of mouse liver panimmunity cell
The method of effective acquisition mouse liver panimmunity cell the following steps are included:
(1) the fresh mouse corpse for taking natural death due to acute hepatic failure is cut after being anticipated completely with the wiping of 75% alcohol swab
Abdomen is opened, mouse liver is isolated and is placed on 10 cm dishes, pays attention to the main blood vessel for retaining gall-bladder and at least 1 centimetre length；
(2) with phosphate buffer (phosphate buffer saline, PBS) the portal vein continuous perfusion, flushing
Liver removes the blood in liver and mouse liver is made to become white from blood red；
(3) gall-bladder and main blood vessel are removed, liver is placed in 10 cm dishes containing PBS and is embathed；
(4) liver is shredded and is placed on containing 4.7 milliliters of improvement Du Shi Eagle's medium (dulbecco ' s
Modified eagle medium, DMEM) and the dissociation pipe of 0.3 milliliter of enzyme mixation in；
(5) dissociation pipe is put into GentleMACSTMDissociator is dissociated in machine, with m_liver_03 mode operation
Twice；
(6) dissociation pipe is placed in 37 DEG C again after, 30 points are digested on the constant-temperature table rotated with 220 revs/min of revolving speed
Clock；
(7) after digesting, then dissociation pipe is placed in and is dissociated in machine, twice with m_liver_04 mode operation；
(8) dissociation manages interior suspension through then being weighed with 5 milliliters of PBS in 100 zut filters to 50 milliliters of centrifuge tubes
Interior residue is managed in outstanding dissociation, is then refiltered to 50 milliliters of centrifuge tube, is finally transferred to 50 milliliters of centrifugation liquid in pipe
In 15 milliliters of centrifuge tube；
(9) at room temperature, 300 relative centrifugal force (g) are centrifuged 10 minutes, pay attention to centrifuge raising speed reduction of speed being adjusted to minimum
Shelves are centrifuged again；
(10) after discarding supernatant, 3 milliliters of 36%Percoll cell separating liquids, 600 relative centrifugal force are added in precipitating
(g) room temperature is centrifuged 15 minutes, notices that centrifuge raising speed reduction of speed is adjusted to deep low gear to be centrifuged again；
(11) supernatant comprising liver cell fragment is discarded, and 2 milliliters of erythrocyte cracked liquid (ACK are added in precipitating
Lysis buffer) to completely remove red blood cell, addition 5ml PBS termination later is split red and is centrifuged relatively 400 within cracking 3 minutes
It is centrifuged 5 minutes under the conditions of 4 DEG C of power (g), pure mouse liver panimmunity cell can be obtained after abandoning supernatant.
In above-mentioned steps:
Phosphate buffer is commercial reagents, and ingredient is sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate and potassium chloride,
Concentration is 0.01 mol/L (0.24 grams per liter of potassium dihydrogen phosphate, 1.42 grams per liter of disodium hydrogen phosphate, 8.0 grams per liter of sodium chloride, chlorination
0.2 grams per liter of potassium), pH value (pH value) 7.4.The configuration method of enzyme mixation are as follows: be added 12 in 100 milliliters of phosphate buffers
Milligram IV, 30 milligram of pronase of clostridiopetidase A and 5 milligrams of deoxyribonuclease Ⅰ powder.Containing 4.7 milliliters of improvement Du Shi she
The dissociation pipe of Ge Er culture medium and 0.3 milliliter of enzyme mixation needs to be placed in 37 DEG C of water-baths and preheats 5 minutes.The filter is: 100
Micron screen.The configuration method of 36%Percoll cell separating liquid are as follows: first by 1 milliliter of 10 times of PBS and 9 milliliter of percoll
Original solution obtains 25 milliliters of 36%Percoll separating liquid after mixing, in the PBS for being added 15 milliliters 1 times.10 times of PBS's
Ingredient is sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate and potassium chloride, and concentration is 0.1 mol/L, pH value (pH value) 7.4.
Cellular morphology observation
1 gained mouse liver panimmunity cell of embodiment is resuspended with 1 milliliter of PBS, and drips at glass slide center, is inverted
Microscopically observation cell.
The identification of immunocyte Wright's staining
(1) immunocyte is resuspended with 1 milliliter of PBS, and drips at a quarter of glass slide full wafer, with slide with equal
Even speed pushes drop to the other end of glass slide, forms cell smear after dry.
(2) 0.5 milliliter of Rui Shi Ji's nurse Sa A liquid is added dropwise and is applying on piece, and dye liquor is allowed to cover entire specimen staining 1 minute.
(3) Rui Shi Ji's nurse Sa B liquid is added on above A liquid to (dripping quantity be A liquid 2-3 times) again, with mouth or ear washing bulb blowout
Gentle breeze makes liquid level generate ripples shape, is sufficiently mixed two liquid, dyes 5 minutes.
(4) (cannot first outwell dye liquor when flushing, should wash away with flowing water, to prevent there is sediment to be deposited on sample) is washed, done
Digital slices scanning means sweeping blade is used after dry, the nucleus of immunocyte can be dyed to aubergine, and endochylema dyes pink.
Isolated immunocyte morphologic observation
Immunocyte is carried out under inverted microscope it has been observed that the cell just separated present it is round, it is full and
Saturating good brightness (20 times of object lens), and the impurity contained is less.
Isolated immunocyte Wright's staining identification
According to scanner scan out come image, it can be observed that the nucleus of isolated immunocyte caught it is purplish red
Color, endochylema has dyed pink, and cell is mostly the lymphocyte of single core, has no red blood cell.
(2) label of mass spectrum streaming antibody
(1) 5 microlitres of final concentration of 2.5 mMs every liter of metal isotopes and polymer attachment are taken；In 37 DEG C of items
Under part, preheat 30 minutes respectively；It is added in the revolving filter of 300 microlitres of R buffer to 50 kilodaltons (kDa)；
(2) it is added in 300 microlitres of the revolving filter of R buffer to 50 kilodaltons (kDa), continuously adds 100
In the antibody of microgram to above-mentioned filter, 12000 relative centrifugal force (g) are centrifuged 10 minutes；
(3) 100ul TCEP reducing agent is added；It preheats to be incubated for for 30 minutes under the conditions of 37 DEG C and goes back original antibody；
(4) it is added in 200 microlitres of the revolving filter of the kilodalton of L-Buffer to 3 (kDa), it is opposite 12,000
Centrifugal force (g) high speed centrifugation 25 minutes；Continue plus 300 microlitres of C-Buffer, 12,000 relative centrifugal force (g) high speed centrifugations
30 minutes；
(5) continue to add in 300 microlitres of C-Buffer to 50 kilodaltons (kDa) revolving filter, 12,000 opposite centrifugations
Power (g) high speed centrifugation 10 minutes；Continue plus 400 microlitres of C-Buffer, 12,000 relative centrifugal force (g) high speed centrifugations 10 divide
Clock；
(6) precipitating in 50 kilodaltons (kDa) revolving filter and in 3 kilodaltons (kDa) revolving filter is mixed
It is even, it is transferred in 50kDa revolving filter, mixes well；It preheats to be incubated for for 90 minutes under the conditions of 37 DEG C and goes back original antibody；
(7) plus in 300 microlitres of W-Buffer to 50 kilodaltons (kDa) filter, 12,000 relative centrifugal force (g) high speed
Centrifugation 10 minutes, three times；
(8) W-buffer recycles the antibody marked, its OD value is measured at 280nm, calculates concentration.
Metal isotope, R buffer, L-Buffer, C-Buffer, W-Buffer reagent being related in above-mentioned steps
Both from MaxPAR X8 antibody coupling kit (Fluidigm, America).
The metal isotope being related in above-mentioned steps are as follows: yttrium (Y-89), indium (In-113, In-115), lanthanum (La-139),
Praseodymium (Pr-141), neodymium (Nd-142, Nd-143, Nd-144, Nd-145, Nd-146, Nd-148, Nd-150), samarium (Sm-147, Sm-
149, Sm-152, Sm-154), europium (Eu-151, Eu-153), gadolinium (Gd-155, Gd-156, Gd-157, Gd-158, Gd-160,
Gd-197), terbium (Tb-159), dysprosium (Dy-161, Dy-162, Dy-163, Dy-164), holmium (Ho-165), erbium (Er-166, Er-
167, Er-168, Er-170), thulium (Tm-169), ytterbium (Yb-171, Yb-172, Yb-173, Yb-174, Yb-176), lutetium (Lu-
175), platinum (Pt-198), bismuth (Bi-209).
The antibody being related in above-mentioned steps are as follows: anti-CD45, anti-CD44, anti-CD19, anti-KI67,
anti-CD24、 anti-MHC II、anti-B220、anti-CD5、anti-CD43、anti-CD38、anti-Ly6G、anti-
Ly6C、 anti-CX3CR1、anti-IgD、anti-CD62L、anti-CD11c、anti-TCRd、anti-CD49a、anti-
CD80、 anti-BST2、anti-CD25、anti-CD3、anti-F4/80、anti-CD115、anti-iNOS、anti-
CXCR3、 anti-CD27、anti-CD103、anti-ICOS、anti-Argnase I、anti-CD49b、anti-Foxp3、
anti-CD127、 anti-CD21、anti-CD23、anti-CD138、anti-CD172a、anti-CTLA-4、anti-
SiglecF、anti-IgM、 anti-CD4、anti-CD8a、anti-CD11b。
Isotope and the case where antibody coupling in above-mentioned steps are as follows:
(3) immunocyte marks
(1) 5 × 10 are taken6The liver panimmunity cell that a step (1) is extracted is added 1 milliliter of FCM analysis and uses and delays
Fliud flushing (FACS Buffer), 400 relative centrifugal force (g) are centrifuged 5 minutes, discard supernatant；
(2) 100 microlitres of phosphate buffers containing metal isotope platinum (Pt-194) are added, and (1:4000, i.e., 0.25 is micro-
Mole every liter) cell is resuspended, it places 5 minutes on ice；
(3) 1 milliliter of FCM analysis is added and is terminated with buffer (FACS Buffer) and react, 400 relative centrifugal force
(g)/5 minute centrifugation, discards supernatant；Antibody confining liquid is added according to volume ratio 1:100, is closed 20 minutes on ice；
(4) be added 50 microlitres coupling metal isotope antibody mixed liquors, on ice be incubated for 30 minutes after, various antibody according to
Certain volume ratio configuration, dilution PBS；
(5) 1 milliliter of FCM analysis is added and is terminated with buffer (FACS Buffer) and react, 400 relative centrifugal force
(g)/5 minute centrifugation, discards supernatant；
(6) 200 microlitres of fixers are added in each sample, overnight；
(7) second days, after 1 milliliter of perm buffer is added into sample, the centrifugation of 800 relative centrifugal force (g)/10 minute,
It discards supernatant
(8) the antibody mixed liquor dyeing of 50 microlitres of coupling metal isotopes for intracellular antigen is added, is incubated on ice
30 minutes；
(9) after 1 milliliter of perm buffer being added, the centrifugation of 800 relative centrifugal force (g)/10 minute is discarded supernatant；
(10) 1 milliliter of FCM analysis is added with after buffer (FACS Buffer), 800 relative centrifugal force (g)/10
Minute centrifugation, discards supernatant, twice；
(11) 1 ml deionized water is added, the centrifugation of 800 relative centrifugal force (g)/10 minute discards supernatant；
(12) cell count is carried out using cell counting board；
(13) 1 ml deionized water is added, the centrifugation of 800 relative centrifugal force (g)/10 minute discards supernatant；
(14) 20%EQ beads water is added to be resuspended, machine in preparation.
It is the cow's serum in every 100 milliliters containing 0.5 gram in above-mentioned steps, in the FCM analysis buffer
The phosphate buffer of albumin (BSA) and 0.02 gram of sodium azide (NaN3)；The formula of antibody confining liquid: every milliliter of phosphate
Contain 20 milligrams of mouse/hamster/rat total IgG in buffer；Antibody for antigen intracellular is anti-KI67, anti-
iNOS,anti-Argnase I,anti-Foxp3,anti-CTLA-4；
In above-mentioned steps, the corresponding antibodies extension rate are as follows:
(4) data are analyzed
It is analyzed using data of the t-SNE and X-shift to mass spectrum streaming, 43 detections of different cell subsets are anti-
For the expression and distribution of body on a thermal map, the expression of unlike signal object and the distribution of different cell subsets pass through viSNE chart
Reveal and.
The liver panimmunity cell of mass spectrum flow cytometer showed label
Due to the antibody identification of metallic element label and the antigen on combination cell surface or inside, marked with metallic element
Antibody cell by one by one be sent into plasmatorch in ionized so that label Metal ion release comes out, release
Metal ion, which is admitted in flight time sensing chamber, carries out separation detection, detector can accurately record that various ions reach when
Between, and then the Precise levels of various metal labels in each cell are conversed, to obtain the antigen table of cell surface or inside
Up to amount.Dimension-reduction treatment is used again, is analyzed with data of the t-SNE and X-shift to mass spectrum streaming, different cell subsets
On a thermal map, the expression of unlike signal object and the distribution of different cell subsets pass through the expression and distribution of 43 detection antibody
ViSNE figure shows.
In Fig. 4, the color placement of the different depths represents the distribution of different cell subsets.In Fig. 5, the color lump of different colours
Represent the expression and distribution situation of 43 antibody of different cell subsets.
Immunocyte divides group
According to the expression of cell surface marker, 33 cell subsets are obtained, are covered in CD45+CD3+T cell, CD45+
CD19+B cell, CD45+CD49a/b+NK cell, CD45+CD11b+CD3-CD19-Myeloid cell.Mouse liver NK cell is main
Including two cell subsets: the liver that expression CD49A does not express CD49B resides NK cell, and expression CD49B does not express CD49A
Traditional NK cell；5 DC cell subsets: thick liquid cell DCs (pDCs), CD103+DCs、CD11b+DCs, cells of monocytic origin
DCs and jejune DCs；Granulocyte is divided into eosinophil and two groups of neutrophil leucocytes.It, will according to the expression of CD172a
Neutrophil cell is divided into CD172a+Neutrophil leucocyte and CD172a-Neutrophil leucocyte.T cell group includes CD4+T- cell, CD8+T-
Cell, CD25+Treg, and gamma delta T cells.B cell is divided into B1 cell subsets and B2 cell subsets.
Claims (10)
1. a kind of method for drafting of acute hepatic failure mouse liver panimmunity cell characteristic map, which is characterized in that including following
Step:
(1) extraction of mouse liver panimmunity cell
The fresh mouse corpse for taking natural death due to acute hepatic failure, wins liver and perfusion goes out its internal blood；By liver
It is dirty shred after half an hour digested with enzyme mixation, then carry out density gradient centrifugation and erythrocyte splitting, obtain pure mouse
Liver panimmunity cell；
(2) label of mass spectrum streaming antibody
Using the MaxPAR X8 antibody coupling kit of Fluidigm company, the U.S., with stable metal isotope and Mouse Liver
Dirty immunocyte surface marker antibody connection, obtains labelled antibody；
(3) immunocyte antibody dyes
Isolated mouse liver panimmunity cell is incubated for labelled antibody, labelled immune cell；
(4) mass spectrum flow cytometer showed
By the mouse liver panimmunity cell after label, machine is analyzed in mass spectrum streaming, using t-SNE and X-shift algorithm to institute
Data are obtained to be analyzed；Then by the expression and distribution of multiple detection antibody of different cell subsets on same thermal map, and
Show the expression of unlike signal object and the distribution of different cell subsets by viSNE chart, mouse liver panimmunity is showed with this
The classification map of cell.
2. the method according to claim 1, wherein the step (1) specifically includes:
(1.1) mouse corpse is wiped with 75% alcohol swab, abdominal cut isolates mouse liver, retains gall-bladder and at least 1 centimetre
The main blood vessel of length；
(1.2) with phosphate buffer the portal vein continuous perfusion, flushing liver, removing internal blood makes liver from blood red
Become white；
(1.3) gall-bladder and main blood vessel are removed, liver is placed in the culture dish containing phosphate buffer and is embathed；
(1.4) liver is shredded to the solution for being placed on and improveing Du Shi Eagle's medium and 0.3 milliliter of enzyme mixation containing 4.7 milliliters
From in pipe；
(1.5) dissociation pipe is put into German MACS company GentleMACSTMDissociator is dissociated in machine, with m_liver_
03 mode operation is twice；
(1.6) dissociation pipe is taken out, is placed on the shaking table of 37 DEG C of constant temperature, 220 revs/min of revolving speeds and digests 30 minutes；
(1.7) after digesting, dissociation pipe is again placed in dissociation machine, twice with m_liver_04 mode operation；
(1.8) will dissociation manage in suspension in 100 zut filters to 50 milliliters of centrifuge tubes, with 5 milliliters of phosphate-buffereds
Liquid is resuspended in dissociation pipe after residue, again filters liquid portion to centrifuge tube；
(1.9) at room temperature, with relative centrifugal force 300g centrifugal treating 10 minutes；
(1.10) after discarding supernatant, 3 milliliters of 36%Percoll cell separating liquids are added into precipitating；At room temperature, with phase
To centrifugal force 600g centrifugal treating 15 minutes；
(1.11) supernatant comprising liver cell fragment is discarded, and 2 milliliters of erythrocyte cracked liquids are added into precipitating and crack 3 minutes
To completely remove red blood cell；Then the termination of addition 5ml phosphate buffer is split red, with relative centrifugal force 400g under the conditions of 4 DEG C
Centrifugal treating 5 minutes；Supernatant is abandoned, pure mouse liver panimmunity cell is obtained.
3. according to the method described in claim 2, it is characterized by: including sodium chloride, di(2-ethylhexyl)phosphate in the phosphate buffer
Hydrogen potassium, disodium hydrogen phosphate and potassium chloride, total concentration are 0.01 mol/L, pH value 7.4.
4. according to the method described in claim 2, it is characterized by: the configuration method of the enzyme mixation are as follows: to 100 milliliters of phosphorus
12 milligrams of clostridiopetidase As, IV, 30 milligram of pronase and 5 milligrams of deoxyribonuclease Ⅰ powder are added in phthalate buffer, mix
It is even.
5. according to the method described in claim 2, it is characterized by: the liver after shredding is placed in dissociation pipe in front of, first
The dissociation pipe of the Du Shi Eagle's medium containing improvement and enzyme mixation is placed in 37 DEG C of water-baths and is preheated 5 minutes.
6. according to the method described in claim 2, it is characterized by: the configuration method of the Percoll cell separating liquid are as follows: first
1 milliliter of 10 times of phosphate buffer is uniformly mixed with 9 milliliters of percoll original solutions, it is slow to add 15 milliliters 1 times of phosphate
Fliud flushing obtains 25 milliliter 36% of Percoll separating liquid.
7. according to the method described in claim 6, it is characterized by: in 10 times of phosphate buffers include sodium chloride,
Potassium dihydrogen phosphate, disodium hydrogen phosphate and potassium chloride, total concentration are 0.1 mol/L, pH value 7.4.
8. the method according to claim 1, wherein the step (1.9), in (1.10) before starting centrifuge,
Centrifuge raising speed reduction of speed first should be adjusted to deep low gear.
9. the method according to claim 1, wherein the step (2) specifically includes: first using metal marker object mark
Remember polymer, obtain the polymer of chelating special metal, be then used for labelled antibody, obtains labelled antibody.
10. the method according to claim 1, wherein in the step (2), the stable metal isotope packet
It includes: Y-89, In-113, In-115, La-139, Pr-141, Nd-142, Nd-143, Nd-144, Nd-145, Nd-146, Nd-
148、Nd-150、Sm-147、Sm-149、Sm-152、Sm-154、Eu-151、Eu-153、Gd-155、Gd-156、Gd-157、Gd-
158、Gd-160、Gd-197、Tb-159、Dy-161、Dy-162、Dy-163、Dy-164、Ho-165、Er-166、Er-167、Er-
168,Er-170,Tm-169,Yb-171,Yb-172,Yb-173,Yb-174,Yb-176,Lu-175,Pt-198,Bi-209；
The antibody has 43, comprising: anti-CD45, anti-CD44, anti-CD19, anti-KI67, anti-CD24,
anti-MHC II、anti-B220、anti-CD5、anti-CD43、anti-CD38、anti-Ly6G、anti-Ly6C、anti-
CX3CR1、anti-IgD、anti-CD62L、anti-CD11c、anti-TCRd、anti-CD49a、anti-CD80、anti-
BST2、anti-CD25、anti-CD3、anti-F4/80、anti-CD115、anti-iNOS、anti-CXCR3、anti-CD27、
anti-CD103、anti-ICOS、anti-Argnase I、anti-CD49b、anti-Foxp3、anti-CD127、anti-
CD21、anti-CD23、anti-CD138、anti-CD172a、anti-CTLA-4、anti-SiglecF、anti-IgM、anti-
CD4、anti-CD8a、anti-CD11b。
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