CN110967321A - Homogeneous system cell fluorescence detection calcium flow method - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 35
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 title claims abstract description 7
- 229910052791 calcium Inorganic materials 0.000 title claims abstract description 7
- 239000011575 calcium Substances 0.000 title claims abstract description 7
- 238000001917 fluorescence detection Methods 0.000 title claims abstract description 5
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 15
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- 210000004027 cell Anatomy 0.000 claims description 51
- 239000000243 solution Substances 0.000 claims description 27
- 238000004458 analytical method Methods 0.000 claims description 22
- 239000000872 buffer Substances 0.000 claims description 12
- 239000011148 porous material Substances 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 10
- 238000011534 incubation Methods 0.000 claims description 9
- 240000001592 Amaranthus caudatus Species 0.000 claims description 8
- 235000009328 Amaranthus caudatus Nutrition 0.000 claims description 8
- 235000012735 amaranth Nutrition 0.000 claims description 8
- 239000004178 amaranth Substances 0.000 claims description 8
- 229940079593 drug Drugs 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 5
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 4
- 239000007995 HEPES buffer Substances 0.000 claims description 4
- QOMNQGZXFYNBNG-UHFFFAOYSA-N acetyloxymethyl 2-[2-[2-[5-[3-(acetyloxymethoxy)-2,7-difluoro-6-oxoxanthen-9-yl]-2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]-n-[2-(acetyloxymethoxy)-2-oxoethyl]-4-methylanilino]acetate Chemical compound CC(=O)OCOC(=O)CN(CC(=O)OCOC(C)=O)C1=CC=C(C)C=C1OCCOC1=CC(C2=C3C=C(F)C(=O)C=C3OC3=CC(OCOC(C)=O)=C(F)C=C32)=CC=C1N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O QOMNQGZXFYNBNG-UHFFFAOYSA-N 0.000 claims description 4
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- 244000309466 calf Species 0.000 claims description 4
- 229920001983 poloxamer Polymers 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- 241000002407 Amaranthus deflexus Species 0.000 claims description 2
- 102000012739 Anion Transport Proteins Human genes 0.000 claims description 2
- 108010079442 Anion Transport Proteins Proteins 0.000 claims description 2
- 238000003650 Calcium Assay Kit Methods 0.000 claims description 2
- 108010078791 Carrier Proteins Proteins 0.000 claims description 2
- 150000007524 organic acids Chemical class 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 239000000523 sample Substances 0.000 claims description 2
- 230000001988 toxicity Effects 0.000 claims description 2
- 231100000419 toxicity Toxicity 0.000 claims description 2
- 238000005259 measurement Methods 0.000 abstract description 10
- 230000008569 process Effects 0.000 abstract description 5
- 238000013537 high throughput screening Methods 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- AIXAANGOTKPUOY-UHFFFAOYSA-N carbachol Chemical compound [Cl-].C[N+](C)(C)CCOC(N)=O AIXAANGOTKPUOY-UHFFFAOYSA-N 0.000 description 4
- 229960004484 carbachol Drugs 0.000 description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- -1 0.0128 nM Chemical compound 0.000 description 2
- OUVXYXNWSVIOSJ-UHFFFAOYSA-N Fluo-4 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=C(C=2)C2=C3C=C(F)C(=O)C=C3OC3=CC(O)=C(F)C=C32)N(CC(O)=O)CC(O)=O)=C1 OUVXYXNWSVIOSJ-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
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- 230000003834 intracellular effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- DBABZHXKTCFAPX-UHFFFAOYSA-N probenecid Chemical compound CCCN(CCC)S(=O)(=O)C1=CC=C(C(O)=O)C=C1 DBABZHXKTCFAPX-UHFFFAOYSA-N 0.000 description 1
- 229960003081 probenecid Drugs 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention relates to the field of cell fluorescence detection, and mainly provides a method which is applicable to FLIPR and does not need to elute extracellular fluorescent substances. In order to realize the aim, the invention provides a method for detecting calcium flow by homogeneous system cell fluorescence by adding a quencher Amaranthh after a cell is loaded with a fluorescent dye. The invention has the following advantages: (1) the cells are not required to replace liquid before measurement; (2) the method is very economical and practical, and the cost of the whole detection process accounts for 10-20% of the cost of the prior art; (3) the whole operation process is simple, and the method can be used for high-throughput screening.
Description
Technical Field
The invention relates to the field of cell fluorescence detection, in particular to a method for detecting calcium flow by homogeneous system cell fluorescence.
Background
High throughput screening is an important link in drug discovery. The exchange of the medium outside the cells is often a time consuming step during the screening assay. Also at this step, loss of cells is an incident that is prone to occur. Addition to the cell medium rather than replacement is generally easier to handle.
Changes in intracellular calcium ions are a widely used and effective indicator of monitoring activation of cell surface receptors. The fluorescent marker Fluo-4 used in this technique changes its fluorescence emission wavelength after binding with calcium ions free in the cells. However, the combination of Fluo-4 and calcium ions remaining outside the cell interferes with intracellular signals. If a quencher is present outside the cell, the extracellular signal is specifically masked, and the experimental process is greatly simplified.
At present, various methods are tried at home and abroad to realize a method without replacing extracellular media, and an economical and practical method for detecting calcium flow by homogeneous system cell fluorescence is urgently needed to be developed.
Disclosure of Invention
The present invention generally provides a method applicable to FLIPR that does not require elution of extracellular fluorescent substances. In order to realize the aim, the invention provides a method for detecting calcium flow by homogeneous system cell fluorescence by adding a quencher Amaranthh after a cell is loaded with a fluorescent dye.
The method comprises the following steps:
a. amaranth powder was weighed and dissolved in FLIPR buffer (1 xHank's buffer +20 mM HEPES, pH 7.4) to give a 25 mg/ml solution;
b. mu.l Fluo-4AM (2 mM) and 10. mu.l pluronic acid (20% in DMSO) were mixed well, added to 10.5 ml FLIPR buffer, 10.5. mu.l calf serum was added,
c. if the cells have an anion pump (organic acid transporter, also called probe (final concentration 2.5 mM) in FLIPR buffer, like CHO cells
d. 100 ul of fluorescent dye solution is added into each well of a 96-well plate, and 100 ul of Amaranth solution is added after the plate is put back into an incubator to be bathed for 0.5h to 48h, which is also different from the prior art (taking F8NW NO Wash Calcium Assay Kit as an example) in order to reduce the toxicity of the quencher Amaranth to cells.
Further, the method also comprises a previous preparation step and a subsequent FLIPR analysis step.
Further, the early preparation steps are as follows: preparing a cell plate: CHO-M5 was plated at a density of 60000 cells per well in 96-well plates (plate type four-week black, clear bottom) and incubated for 16-24 hours.
Further, the early preparation steps are as follows: preparing a cell plate: HEK-M4 cells were seeded at 80000 cells/well in 96-well plates (plate type is black around, clear bottom) and incubated for 16-24 hours.
Further, the subsequent FLIPR analysis steps are as follows: the cells are CHO cells, the culture medium in the pore plate is completely sucked out, 100 mu l of fluorescent dye solution is added, after incubation for 0.5h-48h at 37 ℃, the liquid in the pore plate is sucked out again, 100 mu l of FLIPR buffer solution (containing Amaranth) is added, and then the FLIPR analysis is carried out after the drugs are added according to the experimental requirements.
Preferably, the subsequent FLIPR analysis step is as follows: the cells are CHO cells, the culture medium in the pore plate is completely sucked out, 100 mu l of fluorescent dye solution is added, after incubation for 1h at 37 ℃, the liquid in the pore plate is sucked out again, 100 mu l of FLIPR buffer solution (containing Amararth) is added, and then the FLIPR analysis is carried out after adding the medicine according to the experimental requirements.
Further, the subsequent FLIPR analysis steps are as follows: the cell is HEK cell, 100 mul of fluorescent dye solution is directly added into a cell pore plate, incubation is carried out for 0.5h-48h at 37 ℃, and FLIPR analysis is carried out after the drug is added according to the experimental requirement.
Preferably, the subsequent FLIPR analysis step is as follows: the cells are HEK cells, 100 mu l of fluorescent dye solution is directly added into a cell pore plate, and FLIPR analysis is carried out after incubation for 1h at 37 ℃ and adding drugs according to experimental requirements.
The invention has the following advantages:
(1) the cells are not required to replace liquid before measurement;
(2) the method is very economical and practical, and the cost of the whole detection process accounts for 10-20% of the cost of the prior art;
(3) the whole operation process is simple, and the method can be used for high-throughput screening.
Drawings
FIG. 1 is a general diagram of the FLIPR analysis of CHO-M5; wherein 1-3 are repeated measurement multiple wells (Amaranthh concentration is 0.5 mg/ml), 4-6 are repeated measurement multiple wells (Amaranthh concentration is 1 mg/ml), 7-9 are repeated measurement multiple wells (Amaranthh concentration is 1.5 mg/ml), 10-12 are repeated measurement multiple wells (Amaranthh concentration is 2 mg/ml), and A to H are sequentially increasing concentration carbachol, namely 0.0128 nM, 0.64 nM, 3.2 nM, 16 nM, 80 nM, 400 nM, 2 muM and 10 muM.
FIG. 2 is a general diagram of the analysis of HEK-M4 FLIPR; wherein 1-3 are repeated measurement multiple wells (Amaranthh concentration is 0.5 mg/ml), 4-6 are repeated measurement multiple wells (Amaranthh concentration is 1 mg/ml), 7-9 are repeated measurement multiple wells (Amaranthh concentration is 1.5 mg/ml), 10-12 are repeated measurement multiple wells (Amaranthh concentration is 2 mg/ml), and A to H are sequentially increasing concentration carbachol, namely 0.0128 nM, 0.64 nM, 3.2 nM, 16 nM, 80 nM, 400 nM, 2 muM and 10 muM.
Detailed Description
The following examples will help to understand the present invention, but do not limit the contents thereof.
Example 1
1) Preparing a cell plate: CHO-M5 was seeded at a density of 60000 cells per well in 96-well plates (plate type is black around, transparent at the bottom) and incubated for 16 hours;
2) solution A was prepared by preparing FLIPR buffer (1 xHBSS +20 mM HEPES), then adding probenecid (1M in 1N NaOH) to a final concentration of 2.5 mM, and adjusting pH to 7.4.
3) Preparing a solution B: mu.l Fluo-4AM (2 mM) and 10. mu.l pluronic acid (20% inDMSO) were mixed well.
4) Preparing a fluorescent dye solution: mu.l of solution B was added to 10.5 ml of solution A, and 10.5. mu.l of calf serum was added.
5) Preparing a buffer solution containing Amarath: amaranthh was added to the FLIPR buffer in different volumes to give final concentrations of 2.0 mg/ml, 1.5mg/ml, 1.0mg/ml, 0.5mg/ml, respectively.
6) FLIPR assay: the culture medium in the well plate is completely sucked out, 100 mu l of fluorescent dye solution is added, after incubation for 1h at 37 ℃, the liquid in the well plate is sucked out again, 100 mu l of FLIPR buffer solution (containing Amarath) is added, then the drugs are added according to the experimental requirements, and then FLIPR analysis is carried out, and the result is shown in figure 1.
7) And (4) analyzing results: as shown in FIG. 1, FLIPR signal was enhanced with increasing carbachol concentration on CHO-M5 cells, and Amaranth at different concentrations had no significant effect on FLIPR signal.
Example 2
1) Preparing a cell plate: HEK-M4 cells were seeded at 80000 cells/well in 96-well plates (plate type is black around, clear bottom) and incubated for 24 hours;
2) solution A was prepared by preparing FLIPR buffer (1 xHBSS +20 mM HEPES) and adjusting pH to 7.4.
3) Preparing a solution B: mu.l Fluo-4AM (2 mM) and 10. mu.l pluronic acid (20% inDMSO) were mixed well.
4) Preparing a fluorescent dye solution: mu.l of solution B was added to 10.5 ml of solution A, and 10.5. mu.l of calf serum was added.
5) Preparing a buffer solution containing Amarath: amaranthh was added to the FLIPR buffer in different volumes to give final concentrations of 6.0 mg/ml, 4.5 mg/ml, 3.0 mg/ml, 1.5mg/ml, respectively.
6) FLIPR assay: add 100. mu.l of fluorochrome solution to each well, incubate at 37 ℃ for 1h, add 100. mu.l Amaranthh solution to each well to give Amaranthh final concentrations of 2.0 mg/ml, 1.5mg/ml, 1.0mg/ml, 0.5mg/ml, add drug according to experimental requirements and then carry out FLIPR analysis, the results are shown in FIG. 2.
7) And (4) analyzing results: as shown in fig. 2, FLIPR signal increased with increasing carbachol concentration on HEK-M4 cells, and different concentrations of Amaranth had no significant effect on FLIPR signal.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for homogeneous system cell fluorescence detection of calcium flow is characterized in that: the method comprises the following steps:
a. amaranth powder was weighed and dissolved in FLIPR buffer (1 xHank's buffer +20 mM HEPES, pH 7.4) to give a 25 mg/ml solution;
b. mu.l Fluo-4AM (2 mM) and 10. mu.l pluronic acid (20% in DMSO) were mixed well, added to 10.5 ml FLIPR buffer, 10.5. mu.l calf serum was added,
c. if the cells have an anion pump (organic acid transporter, also called probe (final concentration 2.5 mM) in FLIPR buffer, like CHO cells
d. 100 ul of fluorescent dye solution is added into each well of a 96-well plate, and 100 ul of Amaranth solution is added after the plate is put back into an incubator to be bathed for 0.5h to 48h, which is also different from the prior art (taking F8NW NO Wash Calcium Assay Kit as an example) in order to reduce the toxicity of the quencher Amaranth to cells.
2. The method of claim 1, wherein: also included are a preliminary preparation step and a subsequent FLIPR analysis step.
3. The method of claim 2, wherein: the early preparation steps are as follows: preparing a cell plate: CHO-M5 was plated at a density of 60000 cells per well in 96-well plates (plate type four-week black, clear bottom) and incubated for 16-24 hours.
4. The method of claim 2, wherein: the early preparation steps are as follows: preparing a cell plate: HEK-M4 cells were seeded at 80000 cells/well in 96-well plates (plate type is black around, clear bottom) and incubated for 16-24 hours.
5. The method of claim 2, wherein: the subsequent FLIPR analysis procedure was as follows: the cells are CHO cells, the culture medium in the pore plate is completely sucked out, 100 mu l of fluorescent dye solution is added, after incubation for 0.5h-48h at 37 ℃, the liquid in the pore plate is sucked out again, 100 mu l of FLIPR buffer solution (containing Amaranth) is added, and then the FLIPR analysis is carried out after the drugs are added according to the experimental requirements.
6. The method of claim 5, wherein: the subsequent FLIPR analysis procedure was as follows: the cells are CHO cells, the culture medium in the pore plate is completely sucked out, 100 mu l of fluorescent dye solution is added, after incubation for 1h at 37 ℃, the liquid in the pore plate is sucked out again, 100 mu l of FLIPR buffer solution (containing Amararth) is added, and then the FLIPR analysis is carried out after adding the medicine according to the experimental requirements.
7. The method of claim 2, wherein: the subsequent FLIPR analysis procedure was as follows: the cell is HEK cell, 100 mul of fluorescent dye solution is directly added into a cell pore plate, incubation is carried out for 0.5h-48h at 37 ℃, and FLIPR analysis is carried out after the drug is added according to the experimental requirement.
8. The method of claim 7, wherein: the subsequent FLIPR analysis procedure was as follows: the cells are HEK cells, 100 mu l of fluorescent dye solution is directly added into a cell pore plate, and FLIPR analysis is carried out after incubation for 1h at 37 ℃ and adding drugs according to experimental requirements.
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| PCT/CN2018/118593 WO2020062544A1 (en) | 2018-09-29 | 2018-11-30 | Calcium loss method for cell fluorescence detection of homogeneous system |
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|---|---|---|---|---|
| US20050221426A1 (en) * | 2001-10-26 | 2005-10-06 | Yong Yao | Novel cell-based assays employing voltage and calcium dyes |
| CN101971024A (en) * | 2007-10-15 | 2011-02-09 | 生命技术公司 | Composition and method for measuring thallium influx and efflux |
| CN104350053A (en) * | 2012-06-15 | 2015-02-11 | 大正制药株式会社 | Heteroaromatic methyl cyclic amine derivative |
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| US20050221426A1 (en) * | 2001-10-26 | 2005-10-06 | Yong Yao | Novel cell-based assays employing voltage and calcium dyes |
| CN101971024A (en) * | 2007-10-15 | 2011-02-09 | 生命技术公司 | Composition and method for measuring thallium influx and efflux |
| CN104350053A (en) * | 2012-06-15 | 2015-02-11 | 大正制药株式会社 | Heteroaromatic methyl cyclic amine derivative |
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| TOSHIKATSU OKUMURA等: "Requirement of Intact Disulfide Bonds in Orexin-A Induced Stimulation of Gastric Acid Secretion That Is Mediated by OX1 Receptor Activation", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
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