CN105441417B - A kind of fixing means keeping cell stress balance - Google Patents
A kind of fixing means keeping cell stress balance Download PDFInfo
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- CN105441417B CN105441417B CN201410433243.4A CN201410433243A CN105441417B CN 105441417 B CN105441417 B CN 105441417B CN 201410433243 A CN201410433243 A CN 201410433243A CN 105441417 B CN105441417 B CN 105441417B
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Abstract
The invention discloses a kind of fixing means of holding cell stress balance, take the separation cell for needing fixing process, PBS centrifugal rinsings；PBS is added in the cell mass that supernatant will be abandoned, and cell suspension is coated on culture dish bottom after mixing；Culture dish is placed in 0 DEG C, with 254nm UV lamps with 1.84W/cm2Intensity illumination, accumulated dose 165.6J/cm2；Treated cell is rinsed with PBS and is collected in centrifuge tube；The cell of collection centrifuges 38 minutes, after abandoning supernatant through 260g, be added 4% paraformaldehyde fix 16 seconds after terminate fix.While ensureing cell fixed effect, the stress equilibrium of cell is maintained, keeps the test result in later stage more accurate apparent.It is fixed on the protein denaturation hinge that ensure that for 16 seconds and only make cell membrane surface, and intracellular albumen is in original state, if 4% paraformaldehyde fixes 10 seconds or more, allows for intracellular all albumen all in hinge state.
Description
Technical field
The present invention relates to a kind of fixing means of holding cell stress balance.
Background technology
Cell is the basic unit of life entity structure and vital movement.To eucaryotic cell structure and intracellular chemistry ingredient and biochemistry
The research of effect is the important channel for disclosing life entity physiology and pathologic process.However, different types of active somatic cell is in vitro
Or after being detached from several minutes of its living environment to a few hours, it may appear that autolyzed intracellular structure is broken to degrade with chemical composition.Again
Person, active somatic cell film are a kind of with mobility, the lipid bilayer structure by protein mosaic, the cell of cell interior
Device includes the cytoskeleton for keeping playing an important role with form for the stress structure of cell, is also at the half of gellike shape
In mobile phase endochylema.The behavior of above-mentioned cell causes difficulty with the analysis and research that design feature is cell.For this purpose, scientist establishes
Cure keeping method for cellular prion protein analysis and the cell state of chemical composition functional localization research, i.e. cell is fixed
(fixation).Cell physiological or pathological state can be kept and be made the hardening of tissue by it, convenient for analyzing the making of sample,
Such as pathological section etc..
Currently, scientist uses different fixative and fixing means, is such as used for reach different experiment purposes
Make pathological section formalin fix, the vinegar that the glutaraldehyde for electron microscope analysis or osmic acid are fixed, detected for nucleus
Acid is fixed, the methanol for immunohistochemistry/immunocytochemical assay or paraformaldehyde are fixed, for the second of DNA analysis
Alcohol is fixed and the Carnoy liquid (methanol and glacial acetic acid) for chromosome analysis is fixed etc..But it is suitable for respectively without a kind of
The general fixing means of kind experiment.For traditional cell fixation methods, no matter using which kind of fixative and which kind of step, reason
Think the result is that cell is made fully and completely to be fixed.To obtain this, as a result, fixation usually requires the long period, (dozens of minutes are extremely
A few hours).And according to the present invention is a kind of cell technique for fixing method being different from traditional concept.It is intended that selecting
The set time (only several seconds) of suitable fixative and optimization makes cell keep its existing stress structure and intracellular to be checked
In the state that albumen is non denatured, it is subjected to cell in-situ electrophoresis process, without broken.
Invention content
The purpose of the present invention is just to provide for a kind of fixing means of holding cell stress balance.
To achieve the goals above, the present invention adopts the following technical scheme that：
A kind of fixing means keeping cell stress balance, specifically includes following steps：
(1) the separation cell for needing fixing process is taken, phosphate buffer centrifugal rinsing is added；
(2) phosphate buffer is added in the cell mass that will abandon supernatant, and cell suspension is coated on culture dish bottom after mixing；
(3) culture dish is placed in 0 DEG C, with 254nm UV lamps with 1.84W/cm2Intensity illumination, accumulated dose 165.6J/cm2；
(4) step 3) treated cell phosphate buffer flushing will be passed through to be collected in centrifuge tube；
(5) cell collected centrifuges 3-8 (preferably 5) minutes through 260g, and after abandoning supernatant, the paraformaldehyde for being added 4% is fixed
PBS is added after 1-6 (preferably 3) seconds and terminates fixation.
The pH of PBS used in the present invention is 7.4.
Beneficial effects of the present invention：
First the cell suspension of dispersion is put in before fixed, culture dish is placed in 0 DEG C, with 254nm UV lamps with 1.84W/cm2
Intensity illumination, accumulated dose 165.6J/cm2, strengthen the original state of protein in nucleus and cytoplasmic matrix.
For the cell of collection after centrifugation, the paraformaldehyde for being added 4% fixes 1-6 (preferably 3) second, is utilized in this method
4% paraformaldehyde set time only maintained answering for cell in 1-6 seconds time while ensureing cell fixed effect
Dynamic balance keeps the experimental result in later stage more accurate apparent.
It is fixed on and ensure that within 1-6 seconds that cell is keeping its existing stress structure and waiting for the non denatured shape of Reichl's test into the cell
Under state, it is subjected to cell in-situ electrophoresis process, without broken.If 4% paraformaldehyde fixes 10 seconds or more, cell is allowed for
Interior all albumen are all in hinge state.
Description of the drawings
Fig. 1 is cell in-situ electrophoretic apparatus front view, wherein 1. running buffer liquid pools, 2. filter membranes, 3. side interface channels, 4.
Cell electrophoresis slot；
Fig. 2 is the detection figure that normal human embryonic lung fibroblasts core size influences SSB bonding states, and wherein 2a is shown
RPA32, RPA70 and POT1 testing result；2b and 2c is respectively HFLF in adherent and trypsin digestion suspended state cell
Fluorecence dye figure.
Specific implementation mode
With reference to embodiment, the invention will be further described.
Embodiment 1
Trypsin digestion (detects 3 kinds, respectively to normal human embryonic lung fibroblasts (HFLF) SSBs of culture：
RPA32, RPA70 and POT1) bonding state influence detection：
(1) the normal human embryonic lung fibroblasts cell with 0.25% trypsinization acquisition exponential phase of growth is dense
Degree is 2 × 107HFLF.
(2) cell is primary through phosphate buffer (PBS) centrifugal rinsing, and 200 μ l PBS are added in the cell mass for abandoning supernatant
Afterwards, it is added dropwise in 3.5cm culture dishes bottom, the uniform stall with goods spread out on the ground for sale of the cell suspension of culture dish bottom is distributed in entire training with suction pipette head
Support ware bottom.
(3) culture dish is placed on ice, with 254nm UV lamps with 1.84W/cm2Intensity illumination, accumulated dose 165.6J/cm2。
(4) cell flushing illuminated in culture dish is collected in the centrifuge tube of a 50ml with 20ml PBS respectively.
(5) cell collected centrifuges 5 minutes through 260g, after abandoning supernatant, 4% 200 μ l of paraformaldehyde is added and pre-fix 3 seconds
Clock is added 46ml PBS and terminates fixation.
Embodiment 2
(1) mouse embryo stem cell of exponential phase of growth, cell concentration 2 are obtained with 0.25% trypsinization
×107。
(2) cell is primary through phosphate buffer (PBS) centrifugal rinsing, and 200 μ l PBS are added in the cell mass for abandoning supernatant
Afterwards, it is added dropwise in 3.5cm culture dishes bottom, the uniform stall with goods spread out on the ground for sale of the cell suspension of culture dish bottom is distributed in entire training with suction pipette head
Support ware bottom.
(3) culture dish is placed on ice, with 254nm UV lamps with 1.84W/cm2Intensity illumination, accumulated dose 165.6J/cm2。
(4) cell flushing illuminated in culture dish is collected in the centrifuge tube of a 50ml with 20ml PBS respectively.
(5) cell collected centrifuges 3 minutes through 260g, after abandoning supernatant, 4% 200 μ l of paraformaldehyde is added and pre-fix 1 second
Clock is added 46ml PBS and terminates fixation.
Embodiment 3
(1) tumour cell of exponential phase of growth is obtained with 0.25% trypsinization, cell concentration is 2 × 107。
(2) cell is primary through phosphate buffer (PBS) centrifugal rinsing, and 200 μ l PBS are added in the cell mass for abandoning supernatant
Afterwards, it is added dropwise in 3.5cm culture dishes bottom, the uniform stall with goods spread out on the ground for sale of the cell suspension of culture dish bottom is distributed in entire training with suction pipette head
Support ware bottom.
(3) culture dish is placed on ice, with 254nm UV lamps with 1.84W/cm2Intensity illumination, accumulated dose 165.6J/cm2。
(4) cell flushing illuminated in culture dish is collected in the centrifuge tube of a 50ml with 20ml PBS respectively.
(5) cell collected centrifuges 8 minutes through 260g, after abandoning supernatant, 4% 200 μ l of paraformaldehyde is added and pre-fix 6 seconds
Clock is added 46ml PBS and terminates fixation.
Cell in-situ electrophoresis (cell in situ electrophoresis, CISE), is that cell to be checked is prepared into list
Cell suspension makes its cell individually to dissociate be scattered in the medium of isotonic i.e. stress equilibrium, will be in cell under electric field action
A kind of electrophoretic for being separated from cell of substance.
Cell after embodiment 1 is fixed carries out following experiment：
(1) after 260g is centrifuged 5 minutes and abandoned supernatant, 0.2%Triton X-100200 μ l is added and carry out Cell-transmission model, when
Between 10 minutes, 46ml PBS are added later, 470g is centrifuged 5 minutes, abandons supernatant.
(2) 936 μ l CISE (cell in situ electroporesis) buffer solution (trihydroxy methyl ammonia is added in cell mass
Methylmethane Tris:25mM；Glycine Glycine:192mM；Glucose Glucose:43.2mM；PH8.3), and it is light by pressure-vaccum
Softly cell is disperseed to suspend.
(3) it takes 500 μ l cell suspensions that control of another 50ml centrifuge tube as CISE is added, waits for subsequent bearing
Reason.Remaining cell suspension is used as CISE samples.
(4) CISE devices are assembled, such as see Fig. 1, and connect electrophoresis power.
(5) CISE buffer solutions are added as shown in Figure 1.
(6) power supply is opened, constant voltage mode is set as, adjustment voltage setting to 500V simultaneously starts electrophoresis operation.
(7) rubber or plastic glove are worn, insulation bottom footwear is worn, ensures that human body insulate with ground.With plastic handles pipettor
126 μ l CISE samples are drawn, softly carefully close to liquid level, away from instillation CISE bufferings at cathode side electrophoresis channel opening 1.5cm
Liquid.
(8) sample immediately begins to timing, electrophoresis time 3 minutes after instilling.Timing terminates, and stops power supply.
(9) liquid to be exhausted as possible with pipettor in electrophoresis tank, and come together in liquid is sucked out in a 50ml centrifuge tubes.
(10) CISE buffer solution cleaning down electrophoresis tanks are used, and exhaust Liquid Residue, again electrophoresis tank is noted by step 10 standard
Enter CISE electrophoretic buffers.
(11) step 10) -15 is repeated), until whole CISE samples are disposed by CISE.If last wheel CISE sample
Less than 126 μ l, preceding step operation is still pressed.
(12) liquid measure of CISE samples manifold trunk and CISE control tubes is added to 50ml with CISE buffer solutions respectively.
(13) CISE samples and CISE controls are centrifuged 5 minutes with 1275g rotating speeds, abandons supernatant.
(14) 0.3ml PBS are added in CISE samples and CISE controls respectively, and gently disperse to suspend by cell.
(15) CISE samples and CISE controls are fixed with 70% ethyl alcohol 15ml respectively.
(16) after fixing 24 hours, by CISE samples and CISE controls row flow cytometry detection in the usual way.
Flow cytometry CISE results：
(1) fixed CISE samples and CISE controls are centrifuged 5 minutes with 1275g, abandons supernatant.
(2) 300 μ l PBS are added in CISE sample cells, and suspension cell is divided into trisection, move into three 1.5ml respectively
Eppendorf is managed, and 1ml PBS are added in each Eppendorf pipes respectively, and 560g is centrifuged 5 minutes, abandons supernatant.CISE is compareed
400 μ l PBS are added in pipe, suspension cell is divided into the quartering, four 1.5ml Eppendorf pipes is moved into respectively, respectively every
1ml PBS are added in a Eppendorf pipes, 560g is centrifuged 5 minutes, abandons supernatant.
(3) all Eppendorf pipes are separately added into 3% bovine serum albumin(BSA) that 20 μ l PBS are prepared.
(4) three Eppendorf pipes equipped with CISE samples are marked and are added rabbit-anti RPA32, SANTA CRUZ respectively,
The experiment of final concentration by specification recommended amounts determines), rabbit-anti RPA70 (BETHYL, final concentration by specification recommended amounts experiment determine)
With rabbit-anti POT (abcam, the experiment of final concentration by specification recommended amounts determine) antibody.Equally, by three equipped with CISE controls
Eppendorf pipes mark and rabbit-anti RPA32 (SANTA CRUZ, concentration dose are identical as CISE sample cells), rabbit-anti are added respectively
RPA70 (BETHYL, concentration dose are identical as CISE sample cells) and rabbit-anti POT (abcam, concentration dose and CISE sample cell phases
Antibody together).It is used as antibody morphism pair in addition, 3 μ PBS are added in the 4th Eppendorf pipe equipped with CISE controls and substitute primary antibody
According to (note：It is verified by experiments, is compareed as antibody morphism with the cell of the CISE cells handled and CISE controls, as a result without statistics
Difference is learned, therefore the cell of CISE controls is used to make antibody morphism control).
(5) above-mentioned Eppendorf pipes are incubated at room temperature 2 hours.
(6) above-mentioned Eppendorf pipes are separately added into 1ml PBS, and 500g centrifugal rinsings 2 times abandon supernatant.
(7) all Eppendorf pipes are separately added into 3% bovine serum albumin(BSA) that 20 μ l PBS are prepared.
(8) all Eppendorf pipes are separately added into the goat anti-rabbit igg secondary antibody of 2 μ l FITC labels.Room temperature is protected from light incubation
30 minutes.
(9) above-mentioned Eppendorf pipes are separately added into 1ml PBS, and 500g centrifugal rinsings 2 times abandon supernatant.
(10) by all Eppendorf pipes be separately added into PI (propidium iodide) dye liquor (50 μ g/ml of concentration, routinely DNA contaminate
Liquid method is prepared) 0.5ml, sets 4 DEG C and is protected from light 1 hour.
(11) the conventional two-parameter FCM detections of row (flow cytometer used in the present embodiment is FACSCalibur).DDM patterns
For excluding cell fragment and cell aggregation.Each detection sample obtains 80000 cells.
(12) result is analyzed using Flowjo softwares, as shown in Fig. 2, dotted line compares for antibody morphism, dark solid is
CISE is compareed, and light solid line is CISE samples.Fig. 2 a are respectively RPA32, RPA70 and POT1 testing result from left to right.Such as Fig. 2
It is shown, illustrate the diminution due to nucleus, the bonding state hair of SSBs along Forward after compareing peak compared with CISE after CISE sample peaks
Raw to change, i.e., SSBs is dissociated.Fig. 2 b are the HFLF cell fluorecence dye figures of normal growth, and Fig. 2 c are by trypsase
Postdigestive HFLF cells fluorecence dye figure, 2b, 2c can be seen that HFLF after trypsin digestion, HFLF nucleus hair
It is raw to shrink.
Although the above-mentioned specific implementation mode to the present invention is described, not to the limit of the scope of the present invention
System, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art need not pay
Go out various modifications or changes that creative work can be made still within protection scope of the present invention.
Claims (5)
1. a kind of fixing means keeping cell stress balance, which is characterized in that specifically include following steps：
(1) the separation cell for needing fixing process is taken, phosphate buffer centrifugal rinsing is added；
(2) phosphate buffer is added in the cell mass that will abandon supernatant, and cell suspension is coated on culture dish bottom after mixing；
(3) culture dish is placed in 0 DEG C, with 254nmUV lamps with 1.84W/cm2Intensity illumination, accumulated dose 165.6J/cm2；
(4) step (3) treated cell phosphate buffer flushing will be passed through to be collected in centrifuge tube；
(5) collect cell through 260g centrifuge 3-8 minute, after abandoning supernatant, be added 4% paraformaldehyde fix 1-6 seconds after add
Enter PBS and terminates fixation；
Wherein, step (1) the separation cell is handled with 0.25% trypsinization obtains.
2. keeping the fixing means of cell stress balance as described in claim 1, which is characterized in that step (2) cell
It is added dropwise in 3.5cm culture dishes bottom, the uniform stall with goods spread out on the ground for sale of the cell suspension of culture dish bottom is distributed in entire culture with suction pipette head
Ware bottom.
3. keeping the fixing means of cell stress balance as described in claim 1, which is characterized in that the step (3) will train
Foster ware is placed on ice.
4. keeping the fixing means of cell stress balance as described in claim 1, which is characterized in that the step (5) is collected
Cell be added 4% paraformaldehyde fix 3 seconds.
5. keeping the fixing means of cell stress balance as described in claim 1, which is characterized in that the step (5) is collected
Cell through 260g centrifuge 5 minutes.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410433243.4A CN105441417B (en) | 2014-08-28 | 2014-08-28 | A kind of fixing means keeping cell stress balance |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410433243.4A CN105441417B (en) | 2014-08-28 | 2014-08-28 | A kind of fixing means keeping cell stress balance |
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| Publication Number | Publication Date |
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| CN105441417A CN105441417A (en) | 2016-03-30 |
| CN105441417B true CN105441417B (en) | 2018-09-14 |
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Non-Patent Citations (1)
| Title |
|---|
| paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry(facs) analysis;L.L.LANIER 等;《journal of immunological methods》;19811231;第47卷(第1期);第25-30页 * |
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