CN103983764B - Cell is carried out the methods and applications of labeled in situ - Google Patents
Cell is carried out the methods and applications of labeled in situ Download PDFInfo
- Publication number
- CN103983764B CN103983764B CN201410154914.3A CN201410154914A CN103983764B CN 103983764 B CN103983764 B CN 103983764B CN 201410154914 A CN201410154914 A CN 201410154914A CN 103983764 B CN103983764 B CN 103983764B
- Authority
- CN
- China
- Prior art keywords
- cell
- situ
- carried out
- cholinomimetic
- labeled
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 0 CC*(CC)c(cc1)cc(O2)c1C(c1ccccc1C(OC)=O)=C(C=C1)C2=CC1=*(CC)CC Chemical compound CC*(CC)c(cc1)cc(O2)c1C(c1ccccc1C(OC)=O)=C(C=C1)C2=CC1=*(CC)CC 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
Abstract
The invention provides a kind of method that cell is carried out labeled in situ, comprise the steps: (1) preparation cell culture fluid containing cholinomimetic；(2) cell is carried out labeled in situ.The method that cell carries out labeled in situ provided by the invention first adopts the cell culture fluid containing cholinomimetic to cultivate cell, then follows the trail of the cholinomimetic being marked on cell with reporter molecules.The present invention utilizes on the nitrine of labelling on cholinomimetic and reporter molecules the specific reaction between the dibenzo cyclobutane of labelling that cell is carried out labeled in situ, the method is simple, high-resolution quick, highly sensitive the cholinomimetic on cell membrane, organelle film can not only be positioned and spike, and cytotoxicity is low, and cellular morphology, cytoactive and survival rate is little.Additionally, the invention provides the application in preparing cell in-situ detection kit of a kind of cholinomimetic.
Description
Technical field
The present invention relates to biological technical field, be specifically related to cell is carried out the methods and applications of labeled in situ.
Background technology
Choline (Cho) is the important component part in membrane structure and lipoprotein composition.In eukaryote, the most common group of phospholipid head end is choline, is phospholipid the abundantest in cell membrane containing phosphatidyl choline, is also the principal structural component of cell membrane, plays vital effect in cellular metabolism and signal conduct；AdrianSalic seminar utilizes choline metabolic pathway in cell, by the end modified alkynyl to choline, and by click copper catalysis, the metabolism behavior of choline in cell is carried out tracer analysis with the dyestuff with nitrine.But, nitrine-alkynyl Husigen [3+3] cycloaddition reaction adopted in the method needs copper as catalyst, and the existence of catalyst copper can cause the toxicity of cell, thus is very restricted in the application of living cells or even live body.
Therefore, it is necessary to provide a kind of cytotoxicity is low, affect little cell in-situ tracing method.
Summary of the invention
For solving the problems referred to above, the invention provides a kind of methods and applications that cell is carried out labeled in situ.
React between choline and reporter molecules that the method that cell carries out labeled in situ provided by the invention adopts nitrine to modify and cell is carried out labeled in situ, the method is low to cytotoxicity, and cellular morphology, cytoactive and survival rate is little, it is possible to high-resolution quick, highly sensitive the cholinomimetic on cell membrane is positioned and spike.
First aspect, the invention provides a kind of method that cell is carried out labeled in situ, it is characterised in that comprise the steps:
(1) preparation cell culture fluid containing cholinomimetic
Thering is provided the cell culture fluid without serum, add final concentration of 100 μMs～1000 μMs of cholinomimetic extremely described cholinomimetic, wherein, the structural formula of described cholinomimetic is such as shown in P1:
In formula, k is the natural number of 2～5.
(2) cell is carried out labeled in situ
Take cell to be marked, after adding the cell culture fluid that step (1) processed, be placed in cell culture incubator and cultivate 12～36 hours；Gather in the crops the cell after described cultivation, and after washing with PBS, add the cell culture fluid re-suspended cell without serum, it is subsequently adding reporter molecules to hatch with cell, obtain it be reported that the cell of molecule labeled in situ, wherein, described reporter molecules is the dibenzo cyclobutane molecule that dye molecule is modified.
Unless otherwise noted, cholinomimetic of the present invention is the choline that nitrine is modified, and structural formula is such as shown in described P1.
The present invention adopts culture fluid without serum to cultivate cell, and the culture medium without serum can allow the labelling effect of experimental group more clear, and the culture medium culturing cell growth to cell in 10～40 minutes not containing serum does not have a significant impact.Because in the stage that cell and DBCO-FLlour488 fluorescent dye are hatched altogether, cell is cultivated according to the cell culture medium containing animal serum, owing to animal serum containing abundant nutritional labeling, such as protein, somatomedin, hormone, lipid and mineral etc., these nutritional labelings are likely to make fluorescent dye that the effect reunited to occur, particularly the protein in serum has stronger adsorptivity with a certain amount of electric charge for DBCO-FLlour488 fluorescent dye, thus reducing the contacting efficiency of DBCO-FLlour488 fluorescent dye and nitrine modification choline, the effect making cell in-situ labelling declines to some extent.
The method that cell carries out labeled in situ provided by the invention first adopts the cell culture fluid containing cholinomimetic to cultivate cell, then follows the trail of the cholinomimetic being marked on cell with reporter molecules.The method utilizes on the nitrine of labelling on cholinomimetic and reporter molecules the specific reaction between the dibenzo cyclobutane of labelling that cell is carried out labeled in situ.
Provided by the invention that cell is carried out the method for labeled in situ is low to cytotoxicity and cellular morphology, cytoactive and survival rate is little, it is possible to high-resolution quick, highly sensitive the cholinomimetic on cell membrane is positioned and spike.
It should be noted that in the structural formula that the present invention relates toSymbol for positive charge.
Preferably, described cell to be marked is Human embryo kidney cells, human breast cancer cell, human lung adenocarcinoma cell or Proliferation of Human Ovarian Cell.
Preferably, before the step of the addition cell culture fluid re-suspended cell without serum, the concrete operations of the cell of employing PBS washing results are: adopt PBS washed cell 2～4 times.
Under this optimum condition, the step purpose of employing PBS of the present invention washing be in that to remove not with the cholinomimetic of Cell binding so that between follow-up nitrine and dibenzo cyclobutane, reaction only carries out on the cell containing cholinomimetic.
Preferably, the described employing cell culture fluid re-suspended cell without serum is 1 × 10 to final concentration of cells5～1 × 107/ul。
Preferably, the condition that described reporter molecules and cell carry out hatching is: adding final concentration of 100 μMs～1000 μMs of reporter molecules extremely described reporter molecules, the time hatched is 10～40 minutes.
Preferably, after described reporter molecules and cell are hatched, adopt PBS washed cell 2～4 times.
The step purpose of employing PBS of the present invention washing be in that to remove not with the reporter molecules of Cell binding, it is simple to follow-up carry out fluorescence microscope or laser confocal microscope is observed.
The reporter molecules adopted in the present invention is the molecular activity dyestuff of gained after dibenzo cyclobutane molecule (DBCO) is carried out fluorescent labeling by dye molecule；The structural formula of described reporter molecules is as indicated at a:
Wherein, R is the dye molecule being marked on DBCO, and the atom N of the active group of described dye molecule and DBCO forms covalent bond, thus obtaining described reporter molecules；Adopt the method that DBCO is carried out labelling by dye molecule that common lab labeling method can be adopted to carry out.
Preferably, in described step (2), it is fluorescein, rhodamine, Molecule of Cyanine Dyes, stilbene, naphthalimide, Coumarins, acridine or pyrene class to described dye molecule.
It is pointed out that the dye molecule that the present invention adopts preferably but is not limited to described fluorescein, rhodamine, Molecule of Cyanine Dyes, stilbene, naphthalimide, Coumarins, acridine or pyrene class.
The reporter molecules of gained of the present invention can select different dye molecules to carry out labelling according to the metabolic characteristic of different cells and hobby.
Preferably, described fluorescein is Flour488, Fluorescein isothiocyanate (FITC), hydroxyl fluorescein or Tetrachlorofluorescein..
Concrete, described Flour488 is purchased from sigma company, and article No. is 761621-1MG, and empirical formula's (Xi Er representation) is C32H33N3O8, molecular weight is 587.62, and structural formula is such as shown in B:
Specifically, the structural formula of described FITC is as shown at c:
Preferably, described rhodamine is Rhodamine 101, RB 200 or carboxyl tetramethylrhodamine.
Specifically, the structural formula of described Rhodamine 101 is as shown atd:
Preferably, described Molecule of Cyanine Dyes is thiazole orange, azoles orange or polymethine series cyanine dyes.
It is further preferred that described indole cyanine dyes molecule is Cy3 or Cy5.5.
Specifically, the structural formula of described Cy3 and Cy5.5 is as follows:
Cholinomimetic provided by the invention can pass through the phosphorylation of choline kinase in cell and form the phospholipid containing cholinomimetic, this phospholipid is incorporated on cell membrane, organelle film by a series of intracellular metabolism, thus cell is carried out labeled in situ.Then adopt and provided by the invention reacted with the described cholinomimetic generation click chemistry being combined on cell membrane, organelle film by the DBCO of dye molecule labelling, again through the dye molecule detected on described DBCO, the cholinomimetic on cell membrane, organelle film can be carried out spike.
Coupling reaction between cholinomimetic provided by the invention and report just can quickly occur in room temperature, physiological environment, high-resolution quick, highly sensitive the cholinomimetic on cell membrane, organelle film can be positioned and spike by detecting the dye molecule of described labelling, be one cytokines labeling method very easily.
Second aspect, the invention provides the application in preparing cell in-situ detection kit of a kind of cholinomimetic as described in relation to the first aspect, wherein, described cell is Human embryo kidney cells, human breast cancer cell, human lung adenocarcinoma cell or Proliferation of Human Ovarian Cell, and the structural formula of described cholinomimetic is such as shown in P1:
In formula, k is the natural number of 2～5.
Preferably, described cell in-situ detection kit is for observing the distribution situation under normal or disease conditions of removable cell in animal body.
It is further preferred that described removable cell is lymphocyte or DC cell.
Preferably, the DYNAMIC DISTRIBUTION situation that described cell in-situ detection kit migrated for later stage of malignant cell.
The test kit that the present invention passes through is by the labeled in situ to cell membrane, organelle film, it is thus possible to effectively probe into the pathogeny of disease on cellular level and molecule moisture.
The methods and applications that cell carries out labeled in situ provided by the invention have the advantages that
1) cell in-situ labeling method provided by the invention is low to cytotoxicity, and cellular morphology, cytoactive and survival rate is little, it is possible to high-resolution quick, highly sensitive the cholinomimetic on cell membrane is positioned and spike；
2) coupling reaction between cholinomimetic provided by the invention and reporter molecules just can quickly occur in room temperature, physiological environment, easy to use；
3) cholinomimetic provided by the invention can be used for preparing the in-situ detection reagent box of Human embryo kidney cells, human breast cancer cell, human lung adenocarcinoma cell or Proliferation of Human Ovarian Cell.
Accompanying drawing explanation
The Laser Scanning Confocal Microscope fluorescence shooting figure that Fig. 1 and Fig. 3～6 provide for the embodiment of the present invention；
The result of the fluorescence intensity of the flow cytomery cell that Fig. 2 provides for the embodiment of the present invention.
Detailed description of the invention
The following stated is the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Embodiment 1
The synthesis of a kind of cholinomimetic (AE-Cho), comprises the steps:
1) take glycol dibromide 8.5mL (98.52mmol) and sodium azide 3.2g (49.22mmol) and join in 25mL dimethylformamide (DMF) organic solvent, under 80 DEG C of conditions, stir 20h, be then cooled to room temperature；Subsequently with sodium chloride and ice extraction into heptane three times, take organic layer sodium-chloride water solution and wash three times, by dried over sodium sulfate and carry out concentrating under reduced pressure, it is thus achieved that 1-nitrine-2-bromoethane (yield 81.21%) of purification；
2) take dimethylethanolamine 49.22mmol to add in 15mL oxolane (THF), be subsequently adding the 1-nitrine-2-bromoethane of step (1) purification, when argon, at 0 DEG C of stirring reaction 6h；Reaction obtains the precipitated product of white after terminating, clean three times with ether, vacuum drying, obtains choline (AE-Cho) solid (yield 70.5%) that pure azidoethyl is modified, and the structure of described AE-Cho is such as shown in P2:
The synthesis flow of this AE Cho is as follows:
AE-Cho carries out magnetic resonance detection, and income analysis data are: 1HNMR (600MHz, CD3OD) (VarianVNMR): δ H4.81 (H2O), 4.01 (4H, m), 3.68 (2H, t), 3.58 (2H, m), 3.25 (6H, s) .13CNMR (600MHz, CD3OD): δ C66.33,63.40,55.51,51.65,47.53,44.80.
Embodiment 2
The synthesis of a kind of cholinomimetic (AP-Cho), comprises the steps:
1) take 1,2-dibromopropane 98.52mmol and sodium azide 49.22mmol and join in 25mL dimethylformamide (DMF) organic solvent, under 80 DEG C of conditions, stir 20h, be then cooled to room temperature；Subsequently with sodium chloride and ice extraction into heptane three times, take organic layer sodium-chloride water solution and wash three times, by dried over sodium sulfate and carry out concentrating under reduced pressure, it is thus achieved that 1-nitrine-2-bromoethane (yield 75.59%) of purification；
2) take dimethylethanolamine 49.22mmol to add in 15mL oxolane (THF), be subsequently adding the 1-nitrine-2-N-Propyl Bromide of step (1) purification, when argon, at 0 DEG C of stirring reaction 6h；Reaction obtains the precipitated product of white after terminating, clean three times with ether, vacuum drying, obtains choline (AP-Cho) solid (yield 69.32%) that pure nitrine propyl group is modified, and the structure of described AP-Cho is such as shown in P3:
The synthesis flow of this AP Cho is as follows:
AP-Cho carries out magnetic resonance detection, and income analysis data are: 1HNMR (600MHz, CD3OD): δ H4.82 (H2O), 3.98 (2H, m), 3.50 (6H, m), 3.18 (6H, s), 2.37 (2H, m) .13CNMR (600MHz, CD3OD): δ C65.37,63.93,55.30,51.09,44.78,28.03,22.17.ESI-LC/MS:[M] +=173.1.
Embodiment 3
A kind of method that mankind mastopathy cell (MCF-7) is carried out labeled in situ, comprises the steps:
1) MCF-7: culture fluid is the DMEM culture medium containing 10% calf serum, condition of culture: 37 DEG C, 5%CO2/ 95% air, saturated humidity, change culture fluid every other day, within every 3～4 days, go down to posterity once；
2) cholinomimetic combines at MCF-7 endocellular metabolism: is first washed 3 times by cell with PBS, renews the fresh DMEM culture medium without calf serum；
Arranging experimental group and matched group, wherein, the AE-Cho that embodiment 1 is prepared by experimental group adds cell culture fluid, and matched group adopts the DMEM culture medium without calf serum to replace the AE-Cho of embodiment 1 preparation to add cell culture fluid；
Co-culture 24h, then wash away unnecessary AE-Cho (adopting PBS washed cell 2～4 times) with PBS, add fresh culture medium, be 1 × 10 to final concentration of cells6, standby；
3) reporter molecules (DBCO of this embodiment employing Flour-488 labelling, purchased from sigma company, article No. 761621) MCF-7 mankind mastopathy cell is carried out fluorescent labeling: reporter molecules DBCO-Flour-488 is added in the cell culture fluid that second step obtains, final concentration of 500 μMs of described reporter molecules, hatch 30 minutes, wash away unnecessary reporter molecules (adopting PBS washed cell 2～4 times) with PBS, obtain the cell solution of fluorescent dye labeled in situ；
4) laser confocal microscope (LEICA is adopted, purchased from Hong Kong Jin Yi electronics corporation, brand/model is Leca LEICA/LEICA laser confocal microscope) observe, also adopt nucleus dyestuff Hoechst33258 (purchased from sigma company, article No. is 861405) that the nucleus of experimental group Yu matched group is all dyeed before observation.
The result of gained laser confocal microscope is as it is shown in figure 1, Fig. 1 includes (a), (b), (c), (d), (e) and (A), (B), (C), (D), (E), wherein, (a) in Fig. 1, (b), (c), (d), e result that () is experimental group, (A) in Fig. 1, (B), (C), (D), (E) for the result of matched group, (a) in Fig. 1 and (A) are Fluirescence observation result that DBCO-Flour-488 passage excitation wavelength is 488nm, (b) in Fig. 1 and (B) are Fluirescence observation result that nucleus dyestuff Hoechst33258 passage excitation wavelength is 415nm, the Overlay figure that (c) in Fig. 1 and (C) are Flour-488 passage Yu nucleus dyestuff Hoechst33258 passage observed result, (d) in Fig. 1 and (D) are cell observed result under light field, and (e) in Fig. 1 is (a), (b), (c), the final Overlay figure of observed result of (d), (E) is (A), (B), (C), (D) the final Overlay figure of observed result.
As shown in Figure 1: the cell membrane of experimental group is in green (Fig. 1-a, color does not show, but may determine that cell membrane has fluorescence by observation of cell form), and nucleus is purple (Fig. 1-b, color does not show, but have fluorescence by the known nucleus of observation of cell form), Fig. 1-c is the effect that two kinds of fluorescence of Fig. 1-a and Fig. 1-b excite altogether, and further illustrating the method that MCF-7 cell membrane carries out labeled in situ provided by the invention can carry out labeled in situ to cell membrane；And matched group only has nucleus to be purple (Fig. 1-B, color does not show, but may determine that nucleus has fluorescence by observation of cell form), cell membrane does not have fluorescence (Fig. 1-A, by the known cell membrane unstressed configuration of observation of cell form)；This illustrates that cell membrane can be carried out labeled in situ by the method that MCF-7 cell membrane carries out labeled in situ provided by the invention from the negative；Additionally, from observation of cell form, cell number Fig. 1-d light field it can be seen that relative to matched group, method provided by the invention is without influence on the morphosis of MCF-7 cell, without reducing MCF-7 cytoactive or cell survival rate.
For absolutely proving beneficial effects of the present invention, the present embodiment also adopts flow cytometer that the fluorescence intensity of the cell to fluorescent dye labeled in situ of step of the present invention (3) gained is detected (i.e. experimental group), positive controls and negative control group are set simultaneously, wherein, described positive controls is embodiment 3 step (2) described matched group, the cell of described negative control group is left intact and (need not the culture medium containing cholinomimetic cultivate, also do not hatch altogether with reporter molecules), i.e. the MCF-7 cell of Nostoc commune Vanch.As in figure 2 it is shown, in fig. 2, the result of negative control group, positive controls and experimental group is respectively as shown in curve 1, curve 2 and curve 3, and as shown in Figure 2, the crest of three curves is in diverse location, and experimental procedure is credible for result；It can clearly be seen that, relative to negative control group and positive controls, the fluorescence intensity of experimental group is higher, illustrative experiment group carries out specificity click chemistry reaction due to DBCO-Flour-488 with the AE-Cho being marked on cell membrane and organelle film, so that Flour-488 can carry out labeled in situ on cell membrane and organelle film；Additionally, cell is not done any process by negative control group, being the spontaneous fluorescence of cell itself, positive control is that cell washs, after adding fluorescent dye, the Flour488 fluorescent dye that still can be adsorbed by cell.
Embodiment 4
A kind of method that Human embryo kidney cells (293T) is carried out labeled in situ, comprises the steps:
1) 293T cultivates: culture fluid is 1640 culture medium containing 12% hyclone, condition of culture: 37 DEG C, 5%CO2/ 95% air, saturated humidity, change culture fluid every other day, within every 3～4 days, go down to posterity once；
2) cholinomimetic combines at 293T endocellular metabolism: is first washed 3 times by cell with PBS, renews fresh 1640 culture medium without calf serum；
Arranging experimental group and matched group, wherein, the AE-Cho that embodiment 1 is prepared by experimental group adds cell culture fluid, and matched group adopts 1640 culture medium without calf serum to replace the AE-Cho of embodiment 1 preparation to add cell culture fluid；
Co-culture 12h, then wash away unnecessary AE-Cho (adopting PBS washed cell 2～4 times) with PBS, add fresh culture medium, be 1 × 10 to final concentration of cells5, standby；
3) reporter molecules (DBCO of this embodiment employing Flour-488 labelling, purchased from sigma company, article No. 761621) 293T Human embryo kidney cells is carried out fluorescent labeling: reporter molecules DBCO-Flour-488 is added in the cell culture fluid that second step obtains, final concentration of 100 μMs of described reporter molecules, hatch 10 minutes, wash away unnecessary reporter molecules (adopting PBS washed cell 2～4 times) with PBS, obtain the cell solution of fluorescent dye labeled in situ；
4) laser confocal microscope (LEICA is adopted, purchased from Hong Kong Jin Yi electronics corporation, brand/model is Leca LEICA/LEICA laser confocal microscope) observe, also adopt nucleus dyestuff Hoechst33258 (purchased from sigma company, article No. is 861405) that the nucleus of experimental group Yu matched group is all dyeed before observation.The result of gained laser confocal microscope shooting cell fluorescence signal is as shown in Figure 3, Fig. 3 includes (a), (b), (c), (d), (e) and (A), (B), (C), (D), (E), wherein, (a) in Fig. 3, (b), (c), (d), e result that () is experimental group, (A) in Fig. 3, (B), (C), (D), (E) for the result of matched group, a ()～(e) and (A)～(E) is arranged all in embodiment 3 in Fig. 1 as described in the arranging of (a)～(e) and (A)～(E).
Result shows: the cell membrane of experimental group is in green (Fig. 3-a, color does not show, but may determine that cell membrane has fluorescence by observation of cell form), and nucleus is purple (Fig. 3-b, color does not show, but have fluorescence by the known nucleus of observation of cell form), Fig. 3-c is the effect that two kinds of fluorescence of Fig. 3-a and Fig. 3-b excite altogether, and further illustrating the method that 293T cell membrane carries out labeled in situ provided by the invention can carry out labeled in situ to cell membrane；And matched group only has nucleus to be purple (Fig. 3-B, color does not show, but may determine that nucleus has fluorescence by observation of cell form), cell membrane does not have fluorescence (Fig. 3-A, by the known cell membrane unstressed configuration of observation of cell form)；This illustrates that cell membrane can be carried out labeled in situ by the method that 293T cell membrane carries out labeled in situ provided by the invention from the negative；Additionally, from observation of cell form, cell number Fig. 3-d light field it can be seen that relative to matched group, method provided by the invention is without influence on the morphosis of 293T cell, without reducing 293T cytoactive or cell survival rate.
Embodiment 5
A kind of method that Breast cancer lines (MDA-MB-231) is carried out labeled in situ, comprises the steps:
1) MDA-MB-231 cultivates: culture fluid is the DMEM culture medium containing 12% hyclone, condition of culture: 37 DEG C, 5%CO2/ 95% air, saturated humidity, change culture fluid every other day, within every 3～4 days, go down to posterity once；
2) cholinomimetic combines at MDA-MB-231 endocellular metabolism: is first washed 3 times by cell with PBS, renews the fresh DMEM culture medium without calf serum；
Arranging experimental group and matched group, wherein, the AE-Cho that embodiment 1 is prepared by experimental group adds cell culture fluid, and matched group adopts the DMEM culture medium without calf serum to replace the AE-Cho of embodiment 1 preparation to add cell culture fluid；
Co-culture 36h, then wash away unnecessary AE-Cho (adopting PBS washed cell 2～4 times) with PBS, add fresh culture medium, be 1 × 10 to final concentration of cells7, standby；
3) reporter molecules (DBCO of this embodiment employing Flour-488 labelling, purchased from sigma company, article No. 761621) MDA-MB-231 cell is carried out fluorescent labeling: reporter molecules DBCO-Flour-488 is added in the cell culture fluid that second step obtains, final concentration of 1000 μMs of described reporter molecules, hatch 40 minutes, wash away unnecessary reporter molecules (adopting PBS washed cell 2～4 times) with PBS, obtain the cell solution of fluorescent dye labeled in situ；
4) laser confocal microscope (LEICA is adopted, purchased from Hong Kong Jin Yi electronics corporation, brand/model is Leca LEICA/LEICA laser confocal microscope) observe, also adopt nucleus dyestuff Hoechst33258 (purchased from sigma company, article No. is 861405) that the nucleus of experimental group Yu matched group is all dyeed before observation.The result of gained laser confocal microscope shooting cell fluorescence signal is respectively such as Fig. 4.
Fig. 4 is the laser confocal microscope shooting results of MDA-MB-231 cell, Fig. 4 includes (a), (b), (c), (d), (e) and (A), (B), (C), (D), (E), wherein, (a) in Fig. 4, (b), (c), (d), e result that () is experimental group, (A) in Fig. 4, (B), (C), (D), (E) for the result of matched group, a ()～(e) and (A)～(E) is arranged all in embodiment 3 in Fig. 1 as described in the arranging of (a)～(e) and (A)～(E).
The result of Fig. 4 shows: MDA-MB-231 cell membrane can be carried out labeled in situ by the method that cell membrane carries out labeled in situ provided by the invention；Additionally, from cellular morphology, cell number it can be seen that relative to matched group, method provided by the invention is without influence on the morphosis of MDA-MB-231 cell, without the cytoactive or the cell survival rate that reduce MDA-MB-231 cell.
In addition, the method that present invention also offers the cell in-situ labelling of human lung adenocarcinoma cell (A549), Proliferation of Human Ovarian Cell (SKOV-3), step is in the same manner as in Example 3, except cell culture medium difference (wherein, culture medium respectively DMEM and 1640 that A549 and SKOV-3 adopts), other step is the same.
After obtaining the cell of fluorescent dye labeled in situ of various cell, laser confocal microscope is adopted to observe, the result of gained laser confocal microscope shooting cell fluorescence signal is respectively as shown in Fig. 5～6, wherein, Fig. 5 is the laser confocal microscope shooting results of A549 cell, and Fig. 6 is the laser confocal microscope shooting results of SKOV-3 cell.
Fig. 5 includes (a), (b), (c), (d), (e) and (A), (B), (C), (D), (E), wherein, the result that (a), (b), (c) in Fig. 5, (d), (e) are experimental group, the result that (A), (B), (C) in Fig. 5, (D), (E) are matched group, (a)～(e) and (A)～(E) are arranged all in embodiment 3 in Fig. 1 as described in the arranging of (a)～(e) and (A)～(E).
Fig. 6 includes (a), (b), (c), (d), (e) and (A), (B), (C), (D), (E), wherein, the result that (a), (b), (c) in Fig. 6, (d), (e) are experimental group, the result that (A), (B), (C) in Fig. 6, (D), (E) are matched group, (a)～(e) and (A)～(E) are arranged all in embodiment 3 in Fig. 1 as described in the arranging of (a)～(e) and (A)～(E).
The result of Fig. 5～Fig. 6 shows: A549 and SKOV-3 cell membrane can be carried out labeled in situ by the method that cell membrane carries out labeled in situ provided by the invention；Additionally, from cellular morphology, cell number it can be seen that relative to matched group, method provided by the invention is without influence on the morphosis of A549 and SKOV-3 cell, without the cytoactive or the cell survival rate that reduce by three kinds of cells.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all any amendment, equivalent replacement and improvement etc. made within the spirit and principles in the present invention, should be included within protection scope of the present invention.
Claims (9)
1. the method that cell is carried out labeled in situ, it is characterised in that comprise the steps:
(1) preparation cell culture fluid containing cholinomimetic
Thering is provided the cell culture fluid without serum, add final concentration of 100 μMs～1000 μMs of cholinomimetic extremely described cholinomimetic, wherein, the structural formula of described cholinomimetic is such as shown in P1:
In formula, k is the natural number of 2～5；
(2) cell is carried out labeled in situ
Take cell to be marked, after adding the cell culture fluid that step (1) processed, be placed in cell culture incubator and cultivate 12～36 hours；Gather in the crops the cell after described cultivation, and after washing with PBS, add the cell culture fluid re-suspended cell without serum, it is subsequently adding reporter molecules to hatch with cell, obtain it be reported that the cell of molecule labeled in situ, wherein, described reporter molecules is the diphenyl cyclooctyne molecule that dye molecule is modified；
Described cell to be marked is Human embryo kidney cells, human breast cancer cell, human lung adenocarcinoma cell or Proliferation of Human Ovarian Cell.
2. the method as claimed in claim 1 cell being carried out labeled in situ, it is characterised in that the described employing cell culture fluid re-suspended cell without serum is 1 × 10 to final concentration of cells5～1 × 107Individual/ul.
3. the method as claimed in claim 1 cell being carried out labeled in situ, it is characterized in that, the condition that described reporter molecules and cell carry out hatching is: adding final concentration of 100 μMs～1000 μMs of reporter molecules extremely described reporter molecules, the time hatched is 10～40 minutes.
4. the method as claimed in claim 1 cell being carried out labeled in situ, it is characterised in that after described reporter molecules and cell are hatched, adopts PBS washed cell 2～4 times.
5. the method as claimed in claim 1 cell being carried out labeled in situ, it is characterised in that in described step (2), described dye molecule is fluorescein, Molecule of Cyanine Dyes, stilbene, naphthalimide, acridine or pyrene class.
6. the method as claimed in claim 5 cell being carried out labeled in situ, it is characterised in that described fluorescein is rhodamine or Coumarins.
7. the method as claimed in claim 5 cell being carried out labeled in situ, it is characterised in that described fluorescein is Fluorescein isothiocyanate, hydroxyl fluorescein or Tetrachlorofluorescein..
8. the method as claimed in claim 6 cell being carried out labeled in situ, it is characterised in that described rhodamine is Rhodamine 101, RB 200 or carboxyl tetramethylrhodamine.
9. the method as claimed in claim 5 cell being carried out labeled in situ, it is characterised in that described Molecule of Cyanine Dyes is thiazole orange, azoles orange or polymethine series cyanine dyes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410154914.3A CN103983764B (en) | 2014-04-17 | 2014-04-17 | Cell is carried out the methods and applications of labeled in situ |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410154914.3A CN103983764B (en) | 2014-04-17 | 2014-04-17 | Cell is carried out the methods and applications of labeled in situ |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103983764A CN103983764A (en) | 2014-08-13 |
| CN103983764B true CN103983764B (en) | 2016-06-29 |
Family
ID=51275816
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410154914.3A Active CN103983764B (en) | 2014-04-17 | 2014-04-17 | Cell is carried out the methods and applications of labeled in situ |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103983764B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039242B (en) * | 2015-08-26 | 2018-08-31 | 中国科学院生态环境研究中心 | A method of promoting Ru (II) metal complex cell nuclear uptake |
| CN105693703B (en) * | 2016-01-25 | 2018-12-25 | 济南大学 | A kind of novel Ratiometric fluorescent probe for the imaging of intracellular lysosomal pH |
| EP3587382A1 (en) * | 2018-06-28 | 2020-01-01 | Rheinische Friedrich-Wilhelms-Universität Bonn | Click-mass spectrometry of alkyne-labeled compounds |
| CN110514634B (en) * | 2019-09-02 | 2021-09-21 | 华东理工大学 | Single-cell glycosyl metabolism marking method based on glass nano electrode |
| CN112794813B (en) * | 2020-12-30 | 2022-04-26 | 四川大学华西医院 | Cyclobutene derivative for fluorescent labeling |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101925366A (en) * | 2007-11-21 | 2010-12-22 | 乔治亚大学研究基金公司 | Alkynes and methods of reacting alkynes with 1,3-dipole-functional compounds |
| CN103267850A (en) * | 2013-04-27 | 2013-08-28 | 深圳先进技术研究院 | Probe for pathological diagnosis of tumor, and preparation method and application thereof |
| CN104177451A (en) * | 2013-05-23 | 2014-12-03 | 北京大学 | Non-natural difunctional saccharides, and preparing methods and applications thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010091142A1 (en) * | 2009-02-04 | 2010-08-12 | President And Fellows Of Harvard College | Compositions and methods for labeling and imaging phospholipids |
| US8258347B2 (en) * | 2009-02-19 | 2012-09-04 | University Of Georgia Research Foundation, Inc. | Cyclopropenones and the photochemical generation of cyclic alkynes therefrom |
-
2014
- 2014-04-17 CN CN201410154914.3A patent/CN103983764B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101925366A (en) * | 2007-11-21 | 2010-12-22 | 乔治亚大学研究基金公司 | Alkynes and methods of reacting alkynes with 1,3-dipole-functional compounds |
| CN103267850A (en) * | 2013-04-27 | 2013-08-28 | 深圳先进技术研究院 | Probe for pathological diagnosis of tumor, and preparation method and application thereof |
| CN104177451A (en) * | 2013-05-23 | 2014-12-03 | 北京大学 | Non-natural difunctional saccharides, and preparing methods and applications thereof |
Non-Patent Citations (2)
| Title |
|---|
| "绿色"方法合成可见-近红外发光半导体量子点及其在HBsAg检测中的应用;申怀彬;《中国优秀博士学位论文全文数据库（工程科技I辑）》;20111231;第116页第5.1.8节 * |
| 胆碱的功能化衍生及其在生物标记中的应用;卢贵红;《中国优秀硕士学位论文全文数据库（工程科技I辑）》;20131215(第S1期);正文第5-8页第1.2.3节，第19-25页，第31-36页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103983764A (en) | 2014-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103983764B (en) | Cell is carried out the methods and applications of labeled in situ | |
| CN108514647B (en) | Matrix metalloproteinase-2 specific multi-modal molecular imaging probe, preparation method thereof and application thereof in preparation of tumor imaging agent | |
| Wu et al. | A near-infrared probe for specific imaging of lipid droplets in living cells | |
| Hickey et al. | Fluorescence microscopy—an outline of hardware, biological handling, and fluorophore considerations | |
| CN103030989B (en) | Synthesis method of water-soluble squarylium indocyanine multifunctional cell fluorescent dye | |
| CN106085416A (en) | A kind of aggregation-induced emission nanoparticle and its preparation method and application | |
| CN104164360B (en) | Integrated microfluidic chip and for three-dimensional nodule location, build, recovery method | |
| Wei et al. | Orange-emissive carbon quantum dots for ligand-directed Golgi apparatus-targeting and in vivo imaging | |
| CN104749369A (en) | Fluorescent labeling method for living organisms having cell membrane structures | |
| CN104530102A (en) | Fluorescence copper complexes detecting sulfur ion in vivo and application thereof | |
| Chen et al. | Dinuclear iridium (III) complexes as phosphorescent trackers to monitor mitochondrial dynamics | |
| CN106053405A (en) | A super-resolution optical imaging method based on a single-molecule positioning process | |
| CN104910211A (en) | Cyclometalated iridium (III) complex and preparation method and application thereof in living cell mitochondria dyeing | |
| CN104685051A (en) | Method for culturing a subpopulation of circulating epithelial tumour cells from a body fluid | |
| KR20210013559A (en) | Fluorescently labeled polysaccharide, its manufacturing method and use | |
| CN106117241A (en) | A kind of detect the fluorescent probe of lysosomal pH in cancerous cell | |
| CN104819966A (en) | Method for applying calixarene fluorescent probes to fluorescent imaging of Zn<2+> and F<-> | |
| Wang et al. | A photostable cationic fluorophore for long-term bioimaging | |
| CN103102331B (en) | Pharmaceutical application of chalcone compound containing piperazine ring | |
| CN108276454A (en) | A kind of half sandwich complex of iridium with fluorescent characteristic two tooth cheland containing N^N and preparation method thereof, application | |
| CN107501221A (en) | It is a kind of to fluorescence probe of hydrogen sulfide quick response and its production and use in organism | |
| CN107522773B (en) | Pentapeptide modified rhodamine B compound and preparation method and application thereof | |
| CN105601676A (en) | Ruthenium complex and application thereof | |
| CZ304094B6 (en) | Use of polymethine salts as mitochondrial probes | |
| CN109913206A (en) | A kind of RNA fluorescence probe and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |