CN102288471A - Immunofluorescence staining method for suspension cells - Google Patents
Immunofluorescence staining method for suspension cells Download PDFInfo
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- CN102288471A CN102288471A CN201110211736XA CN201110211736A CN102288471A CN 102288471 A CN102288471 A CN 102288471A CN 201110211736X A CN201110211736X A CN 201110211736XA CN 201110211736 A CN201110211736 A CN 201110211736A CN 102288471 A CN102288471 A CN 102288471A
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Abstract
The invention relates to an immunofluorescence staining method for suspension cells. The immunofluorescence staining method for the suspension cells is characterized by comprising the following steps of: collecting cells into a centrifuge tube; performing 800-gram centrifugation to collect the cells; respectively adding paraformaldehyde to fix, Triton-X100 to pass through and 1 percent bovine serum albumin (BSA) to close; adding a primary antibody and a secondary antibody sequentially to incubate; washing by an 800-gram centrifugation method after the operation of each step; performing diamino phenyl indole (DAPI) staining; and dripping on a glass slide and closing the glass slide to observe. The immunofluorescence staining method for the suspension cells has the advantages that: the immunofluorescence staining process is performed in the centrifuge tube, so the problems that the suspension cells grow on the glass slide difficultly and drop off easily in the test process are solved; the staining result is good; and the method is suitable for the suspension cells and cells which are adhered to the wall difficultly.
Description
Technical field
The present invention relates to a kind of cell dyeing method, specifically, is a kind of immunofluorescence dyeing method of suspension cell.
Background technology
For traditional cellular immunofluorescence dyeing, adopt methods such as cell climbing sheet or direct smear to come pretreatment cell usually, carry out immunofluorescence dyeing again.And for the cell or the cell mass of suspension growth, when adopting cell climbing sheet to handle, regular meeting occur adherent be not in good state or on the slide two sides situations of growth simultaneously all, influence the dyeing and the observation in later stage; In addition, in follow-up dyeing course, the cell that is attached to creep plate comes off easily through after repeatedly washing.
Chinese patent literature CN101329230B discloses a kind of improved method for dyeing immunofluorescence cell, comprise dyeing in fixing/penetrating, penetrating, padding and the nuclear, washing, resuspended totally 5 steps, it is improvement to the Foxp3 method for dyeing immunofluorescence cell of eBioscience company, change two step multiple staining methods into a step multiple staining method, carry out dyeing in cell surface dyeing and the nuclear simultaneously.Chinese patent literature CN101226118 discloses a kind of Cytochemical staining method of compatible with immunofluorescence analysis, relates to Cytochemical staining method of a kind of compatible with immunofluorescence analysis and uses thereof.Chinese patent literature CN102043047A discloses a kind of immunofluorescence method of the fast, economical based on cell climbing sheet.But the immunofluorescence dyeing method about a kind of suspension cell yet there are no report.
Summary of the invention
The objective of the invention is at deficiency of the prior art, a kind of immunofluorescence dyeing method of suspension cell is provided.
For achieving the above object, the technical scheme that the present invention takes is: a kind of immunofluorescence dyeing method of suspension cell, described colouring method may further comprise the steps: first collecting cell is in centrifuge tube, the 800g centrifugal collecting cell, add respectively then that paraformaldehyde (PFA) is fixing, Triton-X100 is penetrating, 1% BSA sealing, add anti-, two anti-hatching again successively, more than all adopt the 800g centrifugation method to wash after the operation of per step, after the last DAPI dyeing, drip to the microslide mounting and observe.
Described colouring method may further comprise the steps:
(total cellular score is 10 for a, collecting cell or maxicell group
6-10
7) in the 2ml centrifuge tube;
B, adding 2ml PBS, gentleness is run up and down and is put, and the centrifugal 5min of 800g abandons supernatant;
C, repetition b step are removed influence of serum in the nutrient culture media;
D, adding 2ml distilled water, gentleness is run up and down and is put, and the centrifugal 5min of 800g abandons supernatant;
E, adding 1ml 4% paraformaldehyde are placed 10min; Flick the pipe truth of a matter time every 2min therebetween, make cell fully contact solution; The centrifugal 5min of 800g abandons supernatant;
F, repetition b, c, d step;
G, the penetrating cell of adding 1ml 0.4% Triton-X-100 are placed 10-20min; Flick the pipe truth of a matter time every 5min therebetween, make cell fully contact solution; The centrifugal 5min of 800g abandons supernatant;
H, repetition b, c, d step;
I, adding 1ml 1% BSA or lowlenthal serum, room temperature is placed 30min, with the heterogenetic antigen on closing cell surface; Flick the pipe truth of a matter time every 5min therebetween, make cell fully contact solution; The centrifugal 5min of 800g abandons supernatant;
J, add one anti-ly, room temperature is placed 2h or 4 ℃ and is spent the night;
K, repetition b, c, d step;
L, lucifuge add fluorescently-labeled two and resist, and room temperature is placed 1h;
M, repetition b, c, d step;
N, adding DAPI and 10ul distilled water, the room temperature lucifuge is placed 5min;
O, cell is dripped to microslide, dry, add the mountant mounting that contains anti-fluorescent quenching agent with pipettor.
Described cell is a suspension cell.
The invention has the advantages that:
The immunofluorescence dyeing method of a kind of suspension cell of the present invention, the process of immunofluorescence dyeing all carries out in centrifuge tube, avoided suspension cell and has been difficult to the problem that creep plate and process of the test come off easily, and coloration result is better, for suspension cell and be difficult to adherent cell, the method is very suitable.
Description of drawings
Accompanying drawing 1 is the fluorescence picture that the EB cell DAPI after RA induces dyes nuclear.
Accompanying drawing 2 is the EB cell fluorescence pictures after RA itself that have a GFP induces.
Accompanying drawing 3 is fluorescence pictures that the EB cell after RA is induced carries out VASA dyeing.
Accompanying drawing 4 is stacks of Fig. 1, Fig. 2, Fig. 3.
Embodiment
Below in conjunction with accompanying drawing embodiment provided by the invention is elaborated.
The Reference numeral and the ingredient that relate in the accompanying drawing are as follows:
Embodiment 1
One, experiment material
Cell: mouse iPS cell (induced pluripotent stem cells, induced multi-potent stem cells), behind the embryoid body (EB) that the Micro-Organism Culture Dish suspension cultured formed in 5 days, add the EB cell of inducing differentiation of vitamin A acid (RA) cultivation after 2 days of 2um.
Reagent: 4% paraformaldehyde, phosphate buffer (Hyclone), 1%BSA(Sigma), TritonX-100, an anti-mouse of anti-VASA rabbit (Abcam), fluorescence two anti-A11072 goat-anti rabbits (Invitrogen), DAPI, mountant (DAKO).
Equipment: low speed centrifuge (Thermo), the low speed shaking table, general refrigerator (beautiful), laser confocal microscope (Leica).
Two, experimental technique
1. collecting RA induces back EB cell mass in the 2ml centrifuge tube;
2. add 2ml PBS, after gentleness was run up and down and put for several times, the centrifugal 5min of 800g abandoned supernatant;
3. repeat 2 steps;
4. add 2ml distilled water, after gentleness was run up and down and put for several times, the centrifugal 5min of 800g abandoned supernatant;
5. add 1ml 4% paraformaldehyde (PFA), place 10min; Flick the pipe truth of a matter time every 2min therebetween, make cell fully contact solution; The centrifugal 5min of 800g abandons supernatant;
6. repeat 2,3,4 steps;
7. add the penetrating cell of 1ml 0.4% Triton-X-100, place 10-20min; Flick the pipe truth of a matter time every 5min therebetween, make cell fully contact solution; The centrifugal 5min of 800g abandons supernatant;
8. repeat 2,3,4 steps;
9. add 1ml 1% BSA, room temperature is placed 30min, flicks the pipe truth of a matter time every 5min therebetween, makes cell fully contact solution; The centrifugal 5min of 800g abandons supernatant;
10. add one anti-(1:100-1:500 dilution), room temperature placement 2h or 4 ℃ spend the night;
Annotate: place the low speed shaking table when spending the night for 4 ℃ and hatch better;
11. repeat 2,3,4 steps;
12. lucifuge adds fluorescently-labeled two anti-(1:100-1:500 dilutions), room temperature is placed 1h;
13. repeat 2,3,4 steps;
14. add DAPI and 10ul distilled water, the room temperature lucifuge is placed 5min;
15. cell is dripped to microslide with pipettor, dry slightly, add the mountant mounting that contains anti-fluorescent quenching agent;
16. laser confocal microscope is observed.
Need to prove that the amount that 4% PFA, 0.4% Triton/X-100,1% BSA add is 1mL, the amount of adding was both enough and cell effect is abundant, can avoid the waste of reagent again.The amount that PBS, distilled water add is 2mL, satisfies the residual reagent of previous step is thoroughly cleaned, and avoids the influence of residual agent.Select centrifugal 5min,, will cause damage by pair cell if centrifugation time is long; If the time is too short, cell can not precipitate fully.The centrifugal 5min of the 800g both injury of pair cell is less, also can be fully centrifugal.
Three, experiment conclusion
Please refer to accompanying drawing 1-4, accompanying drawing 1 is the fluorescence picture that the EB cell DAPI after RA induces dyes nuclear, accompanying drawing 2 is the EB cell fluorescence pictures after RA itself that have a GFP induces, and accompanying drawing 3 is fluorescence pictures that the EB cell after RA is induced carries out VASA dyeing, and accompanying drawing 4 is stacks of Fig. 1, Fig. 2, Fig. 3.Induce expression in the EB cell of differentiation in order to detect reproduction cell mark VASA at RA, the EB cell has been carried out VASA dyeing.The laser co-focusing picture shows that the EB after RA induces expresses VASA, proves that the iPS cell can break up to reproduction cell.
The present invention adopts low-speed centrifugation to suspension cell and be difficult to adherent cell and carry out immunofluorescence dyeing.For suspension cell and be difficult to adherent cell, prior art is the method that is applicable to attached cell by creep plate or smear etc., and for the cell or the cell mass of suspension growth, when adopting cell climbing sheet to handle, the adherent situation that is not in good state or all grows simultaneously on the slide two sides appears in regular meeting, influences the dyeing and the observation in later stage; In addition, in follow-up dyeing course, be attached to creep plate cell through repeatedly the flushing after, come off easily.
The present invention elder generation collecting cell is in the 2ml centrifuge tube, utilize low-speed centrifugal method collecting cell, add respectively then that PFA fixes, Triton-X100 is penetrating, 1% BSA sealing, add anti-, two anti-hatching again successively, more than all adopt the method for low-speed centrifugal to wash after per step operation.After the last DAPI dyeing, drip to the microslide mounting again and observe.The process of immunofluorescence dyeing all carries out in centrifuge tube, avoided suspension cell and has been difficult to the problem that creep plate and process of the test come off easily, and coloration result is better.For suspension cell and be difficult to adherent cell, the method is very suitable.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.
Claims (3)
1. the immunofluorescence dyeing method of a suspension cell, it is characterized in that, described colouring method may further comprise the steps: first collecting cell is in centrifuge tube, the 800g centrifugal collecting cell, add respectively then that paraformaldehyde is fixed, Triton-X100 is penetrating, 1% BSA sealing, add anti-, two anti-hatching again successively, more than all adopt the 800g centrifugation method to wash after per step operation, after the last DAPI dyeing, drip to the microslide mounting and observe.
2. colouring method according to claim 1 is characterized in that, described colouring method may further comprise the steps:
(total cellular score is 10 for a, collecting cell or maxicell group
6-10
7) in the 2ml centrifuge tube;
B, adding 2ml PBS, gentleness is run up and down and is put, and the centrifugal 5min of 800g abandons supernatant;
C, repetition b step are removed influence of serum in the nutrient culture media;
D, adding 2ml distilled water, gentleness is run up and down and is put, and the centrifugal 5min of 800g abandons supernatant;
E, adding 1ml 4% paraformaldehyde are placed 10min; Flick the pipe truth of a matter time every 2min therebetween, make cell fully contact solution; The centrifugal 5min of 800g abandons supernatant;
F, repetition b, c, d step;
G, the penetrating cell of adding 1ml 0.4% Triton-X-100 are placed 10-20min; Flick the pipe truth of a matter time every 5min therebetween, make cell fully contact solution; The centrifugal 5min of 800g abandons supernatant;
H, repetition b, c, d step;
I, adding 1ml 1% BSA or lowlenthal serum, room temperature is placed 30min, with the heterogenetic antigen on closing cell surface; Flick the pipe truth of a matter time every 5min therebetween, make cell fully contact solution; The centrifugal 5min of 800g abandons supernatant;
J, add one anti-ly, room temperature is placed 2h or 4 ℃ and is spent the night;
K, repetition b, c, d step;
L, lucifuge add fluorescently-labeled two and resist, and room temperature is placed 1h;
M, repetition b, c, d step;
N, adding DAPI and 10ul distilled water, the room temperature lucifuge is placed 5min;
O, cell is dripped to microslide, dry, add the mountant mounting that contains anti-fluorescent quenching agent with pipettor.
3. colouring method according to claim 1 is characterized in that, described cell is a suspension cell.
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Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102749243A (en) * | 2012-07-16 | 2012-10-24 | 浙江大学舟山海洋研究中心 | Method for staining nucleuses of shrimp embryos |
| CN103063499A (en) * | 2012-12-03 | 2013-04-24 | 上海海洋大学 | Method for staining actin filaments of suspension cells |
| RU2563679C2 (en) * | 2013-12-11 | 2015-09-20 | Федеральное Государственное Автономное Образовательное Учреждение Высшего Профессионального Образования "Московский Физико-Технический Институт (Государственный Университет)" | Method for immunohistochemical colouring of whole amounts of biological tissue samples (versions) |
| CN105424450A (en) * | 2015-12-17 | 2016-03-23 | 天津医科大学总医院 | Floating sphere immunofluorescent staining method and staining device |
| CN105547791A (en) * | 2015-12-22 | 2016-05-04 | 湖南人康生命科技有限公司 | An immunofluorescent staining method for synapse sites in a nervous system |
| CN105784661A (en) * | 2016-04-06 | 2016-07-20 | 北京雷根生物技术有限公司 | Mounting medium capable of reducing fluorescence decay |
| CN106318974A (en) * | 2016-08-29 | 2017-01-11 | 南方医科大学南方医院 | Cell immobilization technology and AQP4 antibody detection kit prepared through same |
| CN107121547A (en) * | 2016-01-21 | 2017-09-01 | 广东和信健康科技有限公司 | The kit and its application of direct immuno-fluorescent antibody assay enterovirns type 71 and coxsackie virus A 16-type |
| CN107402296A (en) * | 2017-08-22 | 2017-11-28 | 亚能生物技术（深圳）有限公司 | The immunofluorescence dyeing and interpretation method of a kind of circulating tumor cell |
| CN107656044A (en) * | 2017-09-25 | 2018-02-02 | 亚能生物技术（深圳）有限公司 | The detection kit and detection method of a kind of circulating tumor cell |
| CN107884250A (en) * | 2017-11-13 | 2018-04-06 | 深圳博大博聚科技有限公司 | A kind of dye discoloration method based on cell counting count board |
| CN108507849A (en) * | 2018-04-08 | 2018-09-07 | 华中农业大学 | A kind of wheat root nucleus extraction method suitable for immunofluorescence analysis |
| CN110095599A (en) * | 2019-04-22 | 2019-08-06 | 浙江大学 | The Microimmunofluorescence test method of cell-free loss |
| CN110376044A (en) * | 2019-08-29 | 2019-10-25 | 北京银丰鼎诚生物工程技术有限公司 | A kind of method of nerve ball immunofluorescence dyeing |
| CN111175112A (en) * | 2020-01-13 | 2020-05-19 | 浙江卫未生物医药科技有限公司 | Improved microcarrier living cell fluorescent staining method |
| CN111207987A (en) * | 2020-03-13 | 2020-05-29 | 东莞市东阳光生物药研发有限公司 | Immunofluorescence staining method for paraffin-embedded three-dimensional suspension cell mass |
| CN112113820A (en) * | 2020-05-28 | 2020-12-22 | 王剑 | Staining and flaking method of cytopathology sample |
| CN112326539A (en) * | 2020-10-28 | 2021-02-05 | 中国科学院生态环境研究中心 | Quantitative detection method for cell proliferation of whole organ |
| CN113310963A (en) * | 2021-05-31 | 2021-08-27 | 四川大学华西医院 | Improved immunofluorescence detection method for neutrophil NETs |
| WO2021238015A1 (en) * | 2020-05-28 | 2021-12-02 | 王道祥 | Multiple-staining section preparation method for cytopathology sample |
| CN114112605A (en) * | 2020-08-27 | 2022-03-01 | 王剑 | Method for making blood circulation tumor cell pathological chip |
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| CN102749243A (en) * | 2012-07-16 | 2012-10-24 | 浙江大学舟山海洋研究中心 | Method for staining nucleuses of shrimp embryos |
| CN103063499A (en) * | 2012-12-03 | 2013-04-24 | 上海海洋大学 | Method for staining actin filaments of suspension cells |
| RU2563679C2 (en) * | 2013-12-11 | 2015-09-20 | Федеральное Государственное Автономное Образовательное Учреждение Высшего Профессионального Образования "Московский Физико-Технический Институт (Государственный Университет)" | Method for immunohistochemical colouring of whole amounts of biological tissue samples (versions) |
| CN105424450A (en) * | 2015-12-17 | 2016-03-23 | 天津医科大学总医院 | Floating sphere immunofluorescent staining method and staining device |
| CN105424450B (en) * | 2015-12-17 | 2018-12-18 | 天津医科大学总医院 | Suspension cell ball immunofluorescence dyeing method and dyeing apparatus |
| CN105547791B (en) * | 2015-12-22 | 2018-03-02 | 湖南人康生命科技有限公司 | A kind of immunofluorescence dyeing method of nervous system synaptic sites |
| CN105547791A (en) * | 2015-12-22 | 2016-05-04 | 湖南人康生命科技有限公司 | An immunofluorescent staining method for synapse sites in a nervous system |
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| CN105784661A (en) * | 2016-04-06 | 2016-07-20 | 北京雷根生物技术有限公司 | Mounting medium capable of reducing fluorescence decay |
| CN105784661B (en) * | 2016-04-06 | 2021-01-01 | 北京雷根生物技术有限公司 | Sealing piece agent for reducing fluorescence attenuation |
| CN106318974A (en) * | 2016-08-29 | 2017-01-11 | 南方医科大学南方医院 | Cell immobilization technology and AQP4 antibody detection kit prepared through same |
| CN107402296A (en) * | 2017-08-22 | 2017-11-28 | 亚能生物技术（深圳）有限公司 | The immunofluorescence dyeing and interpretation method of a kind of circulating tumor cell |
| CN107656044A (en) * | 2017-09-25 | 2018-02-02 | 亚能生物技术（深圳）有限公司 | The detection kit and detection method of a kind of circulating tumor cell |
| CN107884250A (en) * | 2017-11-13 | 2018-04-06 | 深圳博大博聚科技有限公司 | A kind of dye discoloration method based on cell counting count board |
| CN108507849A (en) * | 2018-04-08 | 2018-09-07 | 华中农业大学 | A kind of wheat root nucleus extraction method suitable for immunofluorescence analysis |
| CN110095599A (en) * | 2019-04-22 | 2019-08-06 | 浙江大学 | The Microimmunofluorescence test method of cell-free loss |
| CN110095599B (en) * | 2019-04-22 | 2021-02-12 | 浙江大学 | Micro-immunofluorescence detection method without cell loss |
| CN110376044A (en) * | 2019-08-29 | 2019-10-25 | 北京银丰鼎诚生物工程技术有限公司 | A kind of method of nerve ball immunofluorescence dyeing |
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| CN111207987A (en) * | 2020-03-13 | 2020-05-29 | 东莞市东阳光生物药研发有限公司 | Immunofluorescence staining method for paraffin-embedded three-dimensional suspension cell mass |
| CN112113820A (en) * | 2020-05-28 | 2020-12-22 | 王剑 | Staining and flaking method of cytopathology sample |
| WO2021238015A1 (en) * | 2020-05-28 | 2021-12-02 | 王道祥 | Multiple-staining section preparation method for cytopathology sample |
| CN114112605A (en) * | 2020-08-27 | 2022-03-01 | 王剑 | Method for making blood circulation tumor cell pathological chip |
| CN112326539A (en) * | 2020-10-28 | 2021-02-05 | 中国科学院生态环境研究中心 | Quantitative detection method for cell proliferation of whole organ |
| CN113310963A (en) * | 2021-05-31 | 2021-08-27 | 四川大学华西医院 | Improved immunofluorescence detection method for neutrophil NETs |
| CN113310963B (en) * | 2021-05-31 | 2023-12-01 | 四川大学华西医院 | Improved neutrophil NETs immunofluorescence detection method |
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