CN102288471B - Immunofluorescence staining method for suspension cells - Google Patents
Immunofluorescence staining method for suspension cells Download PDFInfo
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- CN102288471B CN102288471B CN 201110211736 CN201110211736A CN102288471B CN 102288471 B CN102288471 B CN 102288471B CN 201110211736 CN201110211736 CN 201110211736 CN 201110211736 A CN201110211736 A CN 201110211736A CN 102288471 B CN102288471 B CN 102288471B
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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 immunofluorescent staining, usually adopt the methods such as cell climbing sheet or direct smear to come pretreatment cell, then carry out immunofluorescence dyeing.And for cell or the cell mass of suspension growth, when adopting cell climbing sheet to process, often can occur adherent be not in good state or on the slide two sides situations of growth simultaneously all, affect dyeing and the observation in later stage; In addition, in follow-up dyeing course, be attached to the cell of creep plate through after repeatedly rinsing, easily come off.
Chinese patent literature CN101329230B discloses a kind of improved method for dyeing immunofluorescence cell, comprise dyeing in fixing/penetrating, penetrating, padding and core, washing, resuspended totally 5 steps, it is the 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 simultaneously dyeing in cell surface dyeing and core.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 a kind of immunofluorescence dyeing method about suspension cell yet there are no report.
Summary of the invention
The objective of the invention is for 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 comprises the following steps: first collecting cell is in centrifuge tube, the 800g centrifugal collecting cell, then add respectively that paraformaldehyde (PFA) is fixing, Triton-X100 is penetrating, 1% BSA sealing, add successively again primary antibodie, two anti-hatching, all adopt the centrifugal method of 800g to wash after above per step operation, after last DAPI dyeing, drop to mounting observation on microslide.
Described colouring method comprises the following steps:
(total cellular score is 10 for a, collecting cell or maxicell group
6-10
7) in the 2ml centrifuge tube;
B, add 2ml PBS, gentleness is run up and down and is put, and the centrifugal 5min of 800g abandons supernatant;
C, repetition b step, the impact of removing serum in nutrient culture media;
D, add 2ml distilled water, gentleness is run up and down and is put, and the centrifugal 5min of 800g abandons supernatant;
E, add 1ml 4% paraformaldehyde, 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;
F, repetition b, c, d step;
G, add the 1ml 0.4% penetrating cell of 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;
H, repetition b, c, d step;
I, add 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 primary antibodie, room temperature is placed 2h or 4 ℃ and is spent the night;
K, repetition b, c, d step;
L, lucifuge add fluorescently-labeled two to resist, and room temperature is placed 1h;
M, repetition b, c, d step;
N, add DAPI and 10ul distilled water, the room temperature lucifuge is placed 5min;
O, with pipettor, cell is dropped on microslide, dry, add the mountant mounting that contains anti-fluorescence quenching.
Described cell is 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 is all carried out in centrifuge tube, has avoided suspension cell and has been difficult to creep plate and the caducous problem of process of the test appearance, and coloration result is better, for suspension cell and be difficult to adherent cell, the method is very applicable.
Description of drawings
Accompanying drawing 1 is the fluorescence picture that the EB cell DAPI after RA induces dyes core.
Accompanying drawing 2 is the EB cell fluorescence pictures after RA with GFP own 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 accompanying drawing are as follows:
Embodiment 1
One, experiment material
Cell: mouse iPS cell (induced pluripotent stem cells, induced multi-potent stem cells), after Micro-Organism Culture Dish suspends and cultivates the embryoid body (EB) that 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, the anti-mouse of primary antibodie VASA rabbit (Abcam), the anti-A11072 goat-anti of fluorescence two rabbit (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 rear 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 1ml 0.4% penetrating cell of 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 primary antibodie (1:100-1:500 dilution), room temperature is placed 2h or 4 ℃ and is spent 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. with pipettor, cell is dropped on microslide, slightly dry, add the mountant mounting that contains anti-fluorescence quenching;
16. confocal laser scanning microscope.
Need to prove, the amount that 4% PFA, 0.4% Triton/X-100,1% BSA add is 1mL, and the amount that adds is both enough and cell effect is abundant, can avoid again the waste of reagent.The amount that PBS, distilled water add is 2mL, satisfies the residual reagent of previous step is thoroughly cleaned, and avoids the impact of residual agent.Select centrifugal 5min, if centrifugation time is long, will be to the cell injury; If the time is too short, cell can not precipitate fully.The centrifugal 5min of 800g is both less to the injury of cell, 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 core, accompanying drawing 2 is the EB cell fluorescence pictures after RA with GFP own 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 EB of laser co-focusing Image Display after RA induces expresses VASA, proves that the iPS cell can be to germline.
The present invention adopts low-speed centrifugation to suspension cell and is difficult to adherent cell and carries out immunofluorescence dyeing.For suspension cell and be difficult to adherent cell, prior art is the method for attached cell that is applicable to by creep plate or smear etc., and for cell or the cell mass of suspension growth, when adopting cell climbing sheet to process, often can occur adherent be not in good state or on the slide two sides situations of growth simultaneously all, affect dyeing and the observation in later stage; In addition, in follow-up dyeing course, be attached to creep plate cell through after repeatedly rinsing, easily come off.
The first collecting cell of the present invention is in the 2ml centrifuge tube, utilize low-speed centrifugal method collecting cell, then add respectively that PFA fixes, Triton-X100 is penetrating, 1% BSA sealing, then add successively primary antibodie, two anti-hatching, all adopt the method for low-speed centrifugal to wash after above per step operation.After last DAPI dyeing, then drop to mounting observation on microslide.The process of immunofluorescence dyeing is all carried out in centrifuge tube, has avoided suspension cell and has been difficult to creep plate and the caducous problem of process of the test appearance, and coloration result is better.For suspension cell and be difficult to adherent cell, the method is very applicable.
The above is only the preferred embodiment 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 (1)
1. the immunofluorescence dyeing method of a suspension cell, is characterized in that, described colouring method comprises the following steps:
A, collecting cell add up to 10
6-10
7Cell or maxicell roll into a ball in the 2ml centrifuge tube;
B, add 2ml PBS, gentleness is run up and down and is put, and the centrifugal 5min of 800g abandons supernatant;
C, repetition b step, the impact of removing serum in nutrient culture media;
D, add 2ml distilled water, gentleness is run up and down and is put, and the centrifugal 5min of 800g abandons supernatant;
E, add 1ml 4% paraformaldehyde, 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;
F, repetition b, c, d step;
G, add the 1ml 0.4% penetrating cell of 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;
H, repetition b, c, d step;
I, add 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 primary antibodie, room temperature is placed 2h or 4 ℃ and is spent the night;
K, repetition b, c, d step;
L, lucifuge add fluorescently-labeled two to resist, and room temperature is placed 1h;
M, repetition b, c, d step;
N, add DAPI and 10 μ l distilled water, the room temperature lucifuge is placed 5min;
O, with pipettor, cell is dropped on microslide, dry, add the mountant mounting that contains anti-fluorescence quenching.
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| CN102288471B true CN102288471B (en) | 2013-06-12 |
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| 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 |
| 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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| US6251665B1 (en) * | 1997-02-07 | 2001-06-26 | Cem Cezayirli | Directed maturation of stem cells and production of programmable antigen presenting dentritic cells therefrom |
| JP4411438B2 (en) * | 2005-07-04 | 2010-02-10 | 国立大学法人群馬大学 | Asialo GM1-expressing cell detection reagent, cell detection method and cell classification method using the same, and aging measurement method |
| CN101329230B (en) * | 2008-07-14 | 2010-10-20 | 中国人民解放军第三军医大学 | Improved method for dyeing immunofluorescence cell |
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