CN113533267A - FITC-based cell microenvironment pH determination method and application thereof in osteoclast blocking zone - Google Patents
FITC-based cell microenvironment pH determination method and application thereof in osteoclast blocking zone Download PDFInfo
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Abstract
The invention relates to a cell microenvironment pH determination method based on FITC and application thereof in osteoclast blocking. According to the invention, FITC is combined on the surface to be detected in a chemical modification or physical adsorption mode, and a standard pH control is set, so that the pH of the surface to be detected can be determined through the fluorescence intensity ratio. FITC is used as a fluorescent probe, so that the acquisition is simple and the cost is low; the invention can measure the pH of specific cell microenvironment to realize quantitative analysis of the cell microenvironment.
Description
Technical Field
The invention belongs to the field of biotechnology methods, and particularly relates to a FITC-based cell microenvironment pH determination method and application thereof in osteoclast sealing.
Background
FITC (fluorescein isothiocyanate) is widely used fluorescein, is commonly used for carrying out fluorescent labeling on a secondary antibody in the field of biotechnology so as to realize protein tracing, and is mainly characterized in that N ═ C ═ S groups on FITC and free-NH 2 on protein are subjected to chemical reaction, and then a fluorescent signal can be observed under the excitation of a light source with a specific wavelength.
The pH measuring reagent has been widely researched and applied, and the main mode is that different pH standard substances are utilized to establish a standard curve of the pH measuring reagent in the aspects of fluorescence, absorbance, specific reaction and the like, the numerical value of an object to be measured in the index is measured, and the pH value of the object to be measured is determined through the standard curve.
The cell microenvironment is an important factor influencing the growth and differentiation of cells, wherein the pH is important in the regulation of the cells, and an estimation method for the pH of the cell microenvironment exists at present, but an accurate determination method is not available at present.
The current application of FITC is mainly limited to fluorescent tracing, and the preparation of pH determination reagents has operation thresholds and process complexity. Patent application No. CN201310685086.1 discloses a method for identifying mixed sperm by using fluorescent dyes FITC and MITO, wherein the sperm is labeled by FITC, so that the tracing effect is realized, the application is limited to the fluorescence property of FITC, and the influence of FITC fluorescence from different environmental factors is not discussed, so that the new application is realized. Patent No. CN201510880704.7 discloses a probe method for measuring pH of solution in optical switch type, which automatically synthesizes a fluorescent probe for pH measurement, but the synthesis steps are complicated and the application range is small. None of these patents achieve full utilization of the FITC properties and pH determination for the cellular microenvironment. Therefore, a method for measuring the pH of the cell microenvironment based on FITC is needed, and the pH of the cell microenvironment can be quantitatively analyzed by using simple FITC.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a cell microenvironment pH determination method based on FITC and application thereof in osteoclast blocking. The method can be used for measuring the pH of the cell microenvironment by combining FITC (fluorescein isothiocyanate) on the surface to be measured and measuring the pH of the surface to be measured through the fluorescence intensity ratio, so that the characteristics of the cell microenvironment can be quantitatively analyzed.
A method for measuring the pH value of a cell microenvironment based on FITC comprises the following steps: the FITC is combined on the surface to be detected in a chemical modification or physical adsorption mode, the chemical modification is formed by covalent bonds of an N-C-S group of the FITC and an amino group of the surface to be detected, the physical adsorption mode is that the combination of the FITC on the surface to be detected is realized through charge action, standard pH contrast is set, and the pH of a specific area of the surface to be detected is determined through fluorescence intensity ratio.
Preferably, the standard pH control is selected from a buffer with a known pH.
Preferably, the fluorescence intensity is measured by a fluorescence microscope or a confocal fluorescence microscope.
Preferably, the standard pH control is PBS buffer with known pH.
Compared with the existing pH measuring mode, the method has the remarkable advantages that:
1) FITC is used as a fluorescent probe, so that the method is simple to obtain and low in cost.
2) The pH of a specific cell microenvironment can be measured, and the quantitative analysis of the cell microenvironment is realized.
Therefore, the FITC-based cell microenvironment pH determination method is simple and easy to implement, and the obtained pH value is accurate and has remarkable originality.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 comparison of fluorescence of FITC solution at different pH values.
FIG. 2 Standard Curve of FITC fluorescence intensity vs. pH.
Figure 3 histological validation of pH induced changes in FITC fluorescence.
Figure 4 fluorescent 3D reconstitution of osteoclast-blocked FITC.
FIG. 5 fluorescence intensity of osteoclast blocking zone and FITC at standard pH.
Figure 6 pH calculation of osteoclast block.
Detailed Description
The following examples are provided to illustrate the method for measuring FITC-based pH in a microenvironment of a cell, but they should not be construed as limiting the scope of the present invention.
The preferable scheme of the FITC-based method for measuring pH in a microenvironment of cells according to the present invention is to link FITC to amino groups on the surface of bone through covalent bonds, and measure the osteoclast blocking zone pH through a fluorescence intensity ratio to a standard pH. The specific procedure was as in example 1.
The present invention can also use other binding methods, other pH standards, and other fluorescence measurement methods, all of which can achieve the same technical effects.
Example 1, FITC covalent Cross-Linked bone chips fluorescence intensity was measured using confocal fluorescence microscopy with PBS as the pH standard.
The method comprises the following steps: incubating FITC solution of 0.1M and bone fragments for 1h at 37 ℃;
step two: washing the bone slices with PBS buffer solution with pH of 7.4 for three times;
step three: inoculating BMDM cells to bone fragments according to 10000 per hole, and adding M-CSF and RANKL for induction;
step four: osteoclast formation after 5 days, fixation of cells and nuclear and skeletal staining of cells;
step five: discarding the supernatant, and adding PBS buffer solution with pH of 7.4;
step six: measuring the fluorescence intensity of osteoclast blocking areas and bone fragments in acellular areas by using a confocal fluorescence microscope;
step seven: osteoclast blocking pH was calculated according to a standard curve.
Example 2, FITC covalent Cross-Linked bone chips fluorescence intensity was measured using confocal fluorescence microscopy with NaCl as the pH standard.
The method comprises the following steps: incubating FITC solution of 0.1M and bone fragments for 1h at 37 ℃;
step two: washing the bone slices with PBS buffer solution with pH of 7.4 for three times;
step three: inoculating BMDM cells to bone fragments according to 10000 per hole, and adding M-CSF and RANKL for induction;
step four: osteoclast formation after 5 days, fixation of cells and nuclear and skeletal staining of cells;
step five: discarding the supernatant, and adding physiological saline;
step six: measuring the fluorescence intensity of osteoclast blocking areas and bone fragments in acellular areas by using a confocal fluorescence microscope;
step seven: osteoclast blocking pH was calculated according to a standard curve.
Example 3, FITC physisorption cell slide with PBS as pH standard using ordinary fluorescence microscope determination of fluorescence intensity.
The method comprises the following steps: incubating FITC solution of 0.1M and the cell slide for 1h at 37 ℃;
step two: discarding the supernatant, and adding a culture medium;
step three: inoculating BMDM cells to a climbing tablet according to 10000 per hole, and adding M-CSF and RANKL for induction;
step four: osteoclast formation after 5 days, fixation of cells and nuclear and skeletal staining of cells;
step five: discarding the supernatant, and adding PBS buffer solution with pH of 7.4;
step six: measuring the fluorescence intensity of osteoclast blocking areas and bone fragments in acellular areas respectively by using a fluorescence microscope;
step seven: osteoclast blocking pH was calculated according to a standard curve.
Fluorescence of FITC solution at different pH's in example 1 was compared.
1. Separately adding 0.1M FITC into the cuvette solution with pH of 4-10
2. FITC fluorescence intensity was photographed at different pH using 488nm laser excitation, as shown in FIG. 1.
Example 1 standard curve of FITC fluorescence intensity versus pH.
1. 0.1M FITC was added to each 96-well plate solution at pH 4-10
2. FITC fluorescence intensity at different pH was measured using a 488nm laser excitation with a multifunctional microplate reader and a standard curve was plotted, as shown in FIG. 2.
Histological validation of pH induced changes in FITC fluorescence in example 1.
1. Osteoclast and pH standard regions were observed using a fluorescence microscope, showing that osteoclast blocking causes a dramatic decrease in FITC fluorescence. As shown in fig. 3.
2. Fluorescence pictures were taken and grouped multiple analysis was performed.
Fluorescent 3D reconstitution of osteoclast-blocked FITC in example 1.
1. Osteoclasts and pH standard regions were observed using a fluorescence microscope for 3D reconstruction.
2. Fluorescence intensity curves were plotted for different regions, showing that osteoclast-blocking zone FITC fluorescence was separated from other channels. As shown in fig. 4.
The fluorescence intensity of osteoclast block in example 1 was calculated from FITC at standard pH.
1. 3D reconstruction of osteoclast block and pH standard zones were taken separately.
2. The fluorescence intensity scores of the osteoclast blocking zone and the pH standard zone were calculated. As shown in fig. 5.
The pH of the osteoclast block in example 1 was calculated.
1. And (5) substituting the fluorescence intensity fraction of the pH standard region into a standard curve to obtain the fluorescence intensity.
2. And (4) carrying out fluorescence intensity ratio of the osteoclast blocking zone to the pH standard zone to obtain the osteoclast blocking zone pH. As shown in fig. 6.
The FITC-based cell microenvironment pH determination methods obtained in examples 2 and 3 were respectively subjected to fluorescence comparison of FITC solution at different pH, a standard curve of FITC fluorescence intensity and pH, histological validation of FITC fluorescence change caused by pH, fluorescent 3D reconstruction of osteoclast-blocked FITC, fluorescence intensity calculation of osteoclast-blocked FITC and FITC at standard pH, and pH calculation of osteoclast-blocked. Similar to the results of the FITC-based pH assay for cellular microenvironment in example 1, it is suggested that FITC-based pH assay for cellular microenvironment with similar effects can be achieved by other binding means described above, other pH standards described above, and other fluorometric means described above.
According to the method for measuring the pH value of the cell microenvironment based on the FITC, the FITC is combined on the surface to be measured, the pH value of the surface to be measured is measured through the fluorescence intensity ratio, and the method can be used for measuring the pH value of the cell microenvironment, so that the cell microenvironment characteristics can be quantitatively analyzed.
Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A method for measuring the pH of a cell microenvironment based on FITC is characterized by comprising the following steps: the FITC is combined on the surface to be detected in a chemical modification or physical adsorption mode, the chemical modification is formed by covalent bonds of an N-C-S group of the FITC and an amino group of the surface to be detected, the physical adsorption mode is that the combination of the FITC on the surface to be detected is realized through charge action, standard pH contrast is set, and the pH of a specific area of the surface to be detected is determined through fluorescence intensity ratio.
2. The FITC-based pH assay method for a cellular microenvironment of claim 1, wherein: the standard pH control was selected from buffers with known pH.
3. The FITC-based pH assay method for a cellular microenvironment of claim 1, wherein: the fluorescence intensity is measured by a fluorescence microscope or a confocal fluorescence microscope.
4. The FITC-based pH assay method for a cellular microenvironment of claim 1, wherein:
the method comprises the following steps: incubating FITC solution of 0.1M and bone fragments for 1h at 37 ℃;
step two: washing the bone slices with PBS buffer solution with pH of 7.4 for three times;
step three: inoculating BMDM cells to bone fragments according to 10000 per hole, and adding M-CSF and RANKL for induction;
step four: osteoclast formation after 5 days, fixation of cells and nuclear and skeletal staining of cells;
step five: discarding the supernatant, and adding PBS buffer solution with pH of 7.4;
step six: measuring the fluorescence intensity of osteoclast blocking areas and bone fragments in acellular areas by using a confocal fluorescence microscope;
step seven: osteoclast blocking pH was calculated according to a standard curve.
5. The FITC-based pH assay method for a cellular microenvironment of claim 1, wherein:
the method comprises the following steps: incubating FITC solution of 0.1M and bone fragments for 1h at 37 ℃;
step two: washing the bone slices with PBS buffer solution with pH of 7.4 for three times;
step three: inoculating BMDM cells to bone fragments according to 10000 per hole, and adding M-CSF and RANKL for induction;
step four: osteoclast formation after 5 days, fixation of cells and nuclear and skeletal staining of cells;
step five: discarding the supernatant, and adding physiological saline;
step six: measuring the fluorescence intensity of osteoclast blocking areas and bone fragments in acellular areas by using a confocal fluorescence microscope;
step seven: osteoclast blocking pH was calculated according to a standard curve.
6. The FITC-based pH assay method for a cellular microenvironment of claim 1, wherein:
the method comprises the following steps: incubating FITC solution of 0.1M and the cell slide for 1h at 37 ℃;
step two: discarding the supernatant, and adding a culture medium;
step three: inoculating BMDM cells to a climbing tablet according to 10000 per hole, and adding M-CSF and RANKL for induction;
step four: osteoclast formation after 5 days, fixation of cells and nuclear and skeletal staining of cells;
step five: discarding the supernatant, and adding PBS buffer solution with pH of 7.4;
step six: measuring the fluorescence intensity of osteoclast blocking areas and bone fragments in acellular areas respectively by using a fluorescence microscope;
step seven: osteoclast blocking pH was calculated according to a standard curve.
7. The FITC-based pH assay method for a cellular microenvironment of claim 2, wherein: the standard pH control was PBS buffer with known pH.
8. The application of the FITC-based cell microenvironment pH determination method in osteoclast blocking is characterized in that: an application of a cell microenvironment pH determination method based on FITC in osteoclast blocking area.
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| CN116082450A (en) * | 2022-06-16 | 2023-05-09 | 山东大学 | Near-infrared ratio type pH fluorescent probe, preparation method thereof and application thereof in bone fracture resorption imaging |
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