A method for quantitative analysis of intracellular lipid content based on Bodipy 493 / 503 fluorescent probe

Abstract

This invention provides a method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe, relating to the field of lipid metabolism detection technology. The method includes: (1) determining the absorption spectrum and excitation / emission spectrum of Bodipy 493 / 503 in isopropanol, and determining the maximum absorption wavelength and maximum excitation / emission wavelength of the fluorescent probe in isopropanol based on the spectral results; (2) staining the target cells with the Bodipy 493 / 503 probe after treatment with the compound, observing the changes in fluorescence signal in the field of view before and after isopropanol extraction, and taking pictures; (3) measuring the absorbance and fluorescence intensity of the isopropanol extract, analyzing the experimental results, and comparing them with the results of triglyceride content detection. This quantitative method is simple, fast, sensitive, effective, and has good experimental controllability and repeatability. The analytical results obtained are consistent with those obtained using other lipid content analysis methods, and it can be of great application in the field of research on the lipid metabolism interference effects of chemicals or environmental pollutants.

Description

Technical Field

[0001] This invention relates to the field of lipid metabolism detection technology, specifically to a quantitative analysis method for staining cellular lipids with the Bodipy 493 / 503 probe. Background Technology

[0002] Lipids, as an important class of biomolecules in the body, are one of the basic components of tissues and cells, providing energy and essential fatty acids for cellular metabolism and participating in the regulation of a series of important biological processes. Abnormal lipid metabolism can lead to various metabolic diseases such as hypertriglyceridemia, atherosclerosis, obesity, and metabolic-related fatty liver disease, seriously threatening human health and placing enormous pressure on public health. Therefore, research on the interference of environmental pollutants on lipid metabolism is of significant practical importance.

[0003] In lipid metabolism research, lipophilic dyes are commonly used to analyze lipid content in tissue cells. Commonly used dyes include Oil Red O, Nile Red, and BODIPY 493 / 503. Among them, BODIPY 493 / 503 is a lipophilic green fluorescent probe with maximum excitation and emission wavelengths of 493 nm and 503 nm, respectively. This probe can be used to label the content of neutral lipids in cells, particularly for the localization and analysis of neutral lipid components such as triglycerides in lipid droplets. It can be used for staining and observation of both live and fixed cells. BODIPY 493 / 503 has a high extinction coefficient and fluorescence quantum yield, good photostability, low background value, and is insensitive to changes in solvent pH, making it one of the most widely used fluorescent dyes in lipid metabolism research.

[0004] Due to cellular heterogeneity, the distribution of lipid droplets varies significantly among individual cells, especially after cell treatment with compounds, leading to even more uneven intracellular lipid distribution. Some cell types, such as 3T3-L1 proadipocytes and C3H10T1 / 2 mesenchymal stem cells, possess differentiation capacity. During induced differentiation, the degree of differentiation can differ among different cell types, resulting in uneven lipid droplet distribution in different regions. Analysis using dyes for cell staining combined with microscopic observation and photography is subject to subjective judgment by the experimenter, affecting the accuracy and reliability of the results. To objectively quantify intracellular lipid content, researchers often use Oil Red O staining followed by extraction with organic solvents such as isopropanol, and then measure the absorbance of the extract. However, Oil Red O staining is prone to precipitation, and the dye is easily adsorbed onto well plates, resulting in high staining background. Therefore, the isopropanol extraction method suffers from low sensitivity and poor reproducibility. Current analyses of BODIPY 493 / 503 fluorescent staining primarily rely on photographic acquisition of fluorescence images, followed by software analysis of the fluorescence signals for semi-quantitative analysis. However, this method is also susceptible to the subjectivity of the experimenter. In summary, accurate and reliable quantitative analysis methods for intracellular lipid components based on BODIPY 493 / 503 probe staining are still lacking. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiencies in the prior art and to provide a method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe.

[0006] The specific technical solution adopted in this invention is as follows:

[0007] This invention provides a method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe, comprising the following steps:

[0008] S1: Plotting fluorescence intensity on the ordinate and excitation or emission wavelength on the abscissa, curve fitting was performed to obtain the maximum excitation and emission wavelengths of the Bodipy 493 / 503 fluorescent probe in isopropanol solution at different concentrations.

[0009] The purpose of using different concentrations of Bodipy 493 / 503 fluorescent probes in this invention is to examine whether there are deviations in the maximum absorption wavelength, maximum excitation wavelength, and maximum emission wavelength under different concentration conditions. The results show that the maximum absorption wavelength is the same under different concentration conditions, while the excitation and emission wavelengths show some deviation under different concentration conditions, approximately ±2 nm. Therefore, the effect of concentration can be ignored.

[0010] S2: After plating and culturing the target cells, they were divided into experimental and control groups. Cells in the experimental group were treated with compound exposure. Both experimental and control groups were fixed with cell fixation solution and permeabilized with Triton X-100. Subsequently, under light-protected conditions, cells were analyzed using Bodipy 493 / 503 fluorescent probes and NucBlue. TM Cellular staining agents were used to stain intracellular lipids and the cell nucleus, and finally observed and photographed under a fluorescence microscope;

[0011] Green fluorescent signals (representing lipids) and blue fluorescent signals (representing cell nuclei) can be observed under a fluorescence microscope.

[0012] The only difference between the control and experimental groups was whether or not the cells were exposed to the compound, as the intracellular lipid content changed before and after compound treatment. For example, if the experimental group used an oleic acid solution of dissolved DMSO containing 0.1% DMSO (v / v) during exposure, then the control group would also contain 0.1% DMSO (v / v).

[0013] S3: After photographing, the well plates were dried. Then, 100% isopropanol was added to the cells in the control and experimental groups treated in step S2. After shaking on a shaker for a period of time in the dark, the cell extract was transferred to EP tubes. The residual cells in the well plates after isopropanol extraction were observed and photographed under a fluorescence microscope. Under the fluorescence microscope, the green fluorescence signal in the well plates disappeared.

[0014] The cell extract was then dried and reconstituted with 100% isopropanol. The reconstituted solution was then used to measure the fluorescence intensity and absorbance of the control and experimental groups of cells under the maximum excitation / emission wavelength. By comparing the fluorescence intensity and absorbance of the control and experimental groups of cells, the quantitative analysis of lipid content in the target cells was achieved.

[0015] Preferably, step S1 is as follows:

[0016] Bodipy 493 / 503 fluorescent probes were added to transparent 96-well plates, and the absorbance in the wavelength range of 400 nm to 700 nm was measured. The maximum absorption wavelength was obtained by curve fitting. Bodipy 493 / 503 fluorescent probes were added to 96-well black plates, and the emission wavelength was fixed at 702 nm. The fluorescence intensity after excitation by incident light in the wavelength range of 400 nm to 700 nm was scanned. Bodipy 493 / 503 fluorescent probes were added to 96-well black plates, and the excitation wavelength was fixed at 400 nm. The fluorescence intensity in the wavelength range of 402 nm to 700 nm was scanned. Then, with the fluorescence intensity as the ordinate and the excitation wavelength or emission wavelength as the abscissa, curve fitting was performed to obtain the maximum excitation wavelength and maximum emission wavelength of Bodipy 493 / 503 fluorescent probes in isopropanol solution at different concentrations.

[0017] Preferably, in step S1, the concentrations of the Bodipy 493 / 503 fluorescent probes are 0, 1, 2, 5, 10, 25, and 50 μM, respectively.

[0018] Preferably, in step S1, the maximum absorption wavelength, maximum excitation wavelength, and maximum emission wavelength of the Bodipy 493 / 503 fluorescent probe in isopropanol solution are 496 nm, 493 nm, and 514 nm, respectively.

[0019] Preferably, step S2 is performed in a 6-well plate.

[0020] Preferably, in step S2, if the target cells are HepG2 cells or HK-2 cells, the compound exposure is achieved by treatment with 200 μM oleic acid (more preferably, the test is performed after 24 hours of treatment); if the target cells are 3T3-L1 or C3H10T1 / 2 cells, the compound exposure is achieved by adding 2 μM rosiglitazone at the beginning of cell differentiation (more preferably, the test is performed after 8 days of treatment).

[0021] Preferably, in step S2, the concentration of the Bodipy 493 / 503 fluorescent probe is 10 μM, the volume is 0.5 mL, and the staining time is 30 min; NucBlue... TM The nuclear staining agent was prepared by diluting with PBS buffer, and 2 drops of raw NucBlue were added to each milliliter of PBS buffer. TM The staining agent was used, and the staining time was 15 minutes.

[0022] Preferably, in step S3, the volume of isopropanol added for the first time is 0.3 mL, and the extraction time is 5 min; the volume of isopropanol added during reconstitution is 0.1 mL.

[0023] Preferably, in step S3, the reconstituted solution is taken into a 96-well plate to measure the cell fluorescence intensity and absorbance.

[0024] Preferably, in step S3, the volume of the reconstituted solution added is 45 μL, the absorbance detection wavelength is 496 nm, and the fluorescence detection excitation and emission wavelengths are 493 nm and 514 nm, respectively.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] This invention utilizes isopropanol to dissolve Bodipy 493 / 503 fluorescent dye and analyzes its spectral characteristics in isopropanol to obtain its maximum absorption wavelength and maximum excitation / emission wavelength. For cells stained with the Bodipy 493 / 503 fluorescent probe, isopropanol is used for extraction, and the absorbance and fluorescence intensity of the extract are analyzed to achieve quantitative analysis of intracellular Bodipy 493 / 503. By comparing the fluorescence intensity and absorbance of the extracts from the experimental and control groups, the effect of compound treatment on cellular lipid content is demonstrated. The quantitative method of this invention is simple, rapid, sensitive, effective, and exhibits good experimental controllability and reproducibility. The analytical results obtained are consistent with those obtained using other lipid content analysis methods. Attached Figure Description

[0027] Figure 1 The absorption spectrum (A) of Bodipy 493 / 503 in isopropanol in Example 1 of the present invention, and the absorbance standard curve (B) of different concentrations of probe at their maximum absorption wavelength;

[0028] Figure 2 The images show the excitation (A) and emission (B) spectra of Bodipy 493 / 503 in isopropanol in Example 1 of this invention, as well as the fluorescence standard curves (C) of different concentrations of probe at excitation / emission wavelengths of 493 nm and 514 nm.

[0029] Figure 3 The images show fluorescence images of HepG2 cells before and after extraction with isopropanol after Bodipy 493 / 503 staining in Example 1 of this invention.

[0030] Figure 4 The image shows the fluorescence intensity (A) and absorbance (B) of Body 493 / 50 extracted with isopropanol in HepG2 cells in Example 1 of this invention, and a comparison (C) of the results with the intracellular triglyceride content detection.

[0031] Figure 5 The images shown are fluorescence images of C3H1071 / 2 cells before and after extraction with isopropanol after Bodipy 493 / 503 staining in Example 2 of this invention.

[0032] Figure 6 The image shows the fluorescence intensity (A) and absorbance (B) of Body 493 / 50 extracted with isopropanol in C3H1071 / 2 cells in Example 2 of this invention, and a comparison (C) with the results of intracellular triglyceride content detection. Detailed Implementation

[0033] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0034] This invention relates to a method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe. The method mainly includes the following steps: First, the absorption and excitation / emission spectra of Bodipy 493 / 503 in isopropanol are determined. Based on the spectral results, the maximum absorption wavelength and maximum excitation / emission wavelength of the fluorescent probe in isopropanol are determined. Target cells treated with the compound serve as the experimental group. The cells are stained with the Bodipy 493 / 503 probe, and the changes in cell fluorescence signals in the wells before and after isopropanol extraction are observed and photographed. The absorbance and fluorescence intensity of the isopropanol extracts in the experimental group and the control group (solvent control group) are measured, and the experimental results are analyzed to determine the effect of compound treatment on intracellular lipid content. The results obtained by the method of this invention are compared with the results of triglyceride content detection in existing technologies to verify the accuracy of the method of this invention.

[0035] The method of the present invention will be described in detail below with reference to specific embodiments.

[0036] Example 1

[0037] This embodiment uses HepG2 cells as the target cells and employs the Bodipy 493 / 503 fluorescent probe to quantitatively analyze intracellular lipid content. The specific steps are as follows:

[0038] S1: Spectroscopic characterization of Bodipy 493 / 503 in isopropanol

[0039] The instruments used in this step are: a full-wavelength multi-mode microplate reader, a vortex mixer, and pipettes; materials include: black 96-well plates, clear 96-well plates, 1.5 mL centrifuge tubes, and pipette tips. Reagents include: Bodipy 493 / 503 (TICEL), and isopropanol.

[0040] (1) Characterization of Bodipy 493 / 503 UV-Vis absorption spectrum

[0041] 1. Weigh 26.2 mg of Bodipy 493 / 503 and add it to 1 mL of isopropanol to prepare a 100 mM Bodipy 493 / 503 stock solution. Mix well and set aside.

[0042] 2. Serially dilute the 100mM Bodipy 493 / 503 stock solution to prepare 0, 1, 2, 5, 10, 25, and 50 μM Bodipy 493 / 503 working solutions. Add 50 μL of each concentration of Bodipy 493 / 503 working solution to a 96-well transparent plate with flat bottom round wells, and read the absorbance of each well in the range of 300-700 nm using a microplate reader.

[0043] 3. Using Graphpad Prism 9, the wavelength corresponding to the maximum absorption peak was obtained. Curve fitting was then performed based on the maximum absorption peak values ​​at different concentrations to obtain a standard curve. The results are shown below. Figure 1 As shown in the figure, the maximum absorption wavelength of Bodipy493 / 503 in isopropanol is 496 nm, and the fluorescence intensity at this wavelength shows a good linear relationship with the dye concentration within the detection concentration range.

[0044] (2) Characterization of excitation and emission spectra of Bodipy 493 / 503

[0045] 1. Under light-protected conditions, 50 μL of different concentrations of Bodipy 493 / 503 working solution were added to 96-well microplates with flat bottoms. The emission wavelength was fixed at 702 nm. The fluorescence intensity of each well was read using a microplate reader at different excitation wavelengths within the range of 200-700 nm. Curve fitting was performed using Graphpad Prism 9 to obtain the excitation spectra at different concentrations, and the maximum excitation wavelength corresponding to the peak values ​​was determined. The results are shown below. Figure 2 As shown in (A), the maximum excitation wavelength of Bodipy 493 / 503 in isopropanol is 493 nm.

[0046] 2. Under light-protected conditions, 50 μL of different concentrations of Bodipy 493 / 503 working solution were added to 96-well microplates with flat bottoms. The excitation wavelength was fixed at 400 nm. The fluorescence intensity of each well was read using a microplate reader at different excitation wavelengths within the range of 402-700 nm. Curve fitting was performed using Graphpad Prism 9 to obtain the emission spectra at different concentrations, and the maximum emission wavelength corresponding to the peak values ​​was determined. The results are shown below. Figure 2 As shown in (B), the maximum emission wavelength of Bodipy 493 / 503 in isopropanol is 514 nm.

[0047] The fluorescence intensity at the maximum excitation wavelength of 493 nm and the maximum emission wavelength of 514 nm was fitted with the corresponding concentrations to obtain a standard curve, as shown in the figure. Figure 2 As shown in (C), the fluorescence intensity under this excitation / emission wavelength condition exhibits a good linear relationship with the corresponding concentration within the detection concentration range.

[0048] S2: HepG2 cell culture and oleic acid-induced lipid accumulation

[0049] The instruments used in this step are: a full-wavelength multi-functional microplate reader, a carbon dioxide cell culture incubator, an inverted fluorescence microscope, a metal bath, a vortex mixer, and pipettes. Materials: HepG2 cells, 6-well plates, clear 96-well plates, 1.5 mL centrifuge tubes, and pipette tips. Reagents: DMEM high-glucose medium (containing phenol red), penicillin / streptomycin, fetal bovine serum (Gibco), oleic acid (Sigma), Triton X-100 (Solepro), DMSO (Sigma), phosphate-buffered saline (PBS), isopropanol, Bodipy 493 / 503, and NucBlue. TM Invitrogen, a cell nucleus fixation staining agent TM Cell tissue fixative (Solepro), triglyceride assay kit (Pulley).

[0050] After being revived and passaged, HepG2 cells were cultured at 5 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 10 cells / well in 6-well plates. After 24 hours, the cells were divided into control and experimental groups. Cells in the experimental group were exposed to 200 μM oleic acid (OA), while those in the control group were treated with 0.1% DMSO (v / v). After 24 hours of exposure, the culture medium was discarded, the cells were washed twice with PBS, fixed with 1 mL of cell fixative for 1 hour, and then 1 mL of 0.1% Triton X-100 (v / v) was added to increase cell membrane permeability. After 15 minutes, 0.5 mL of 10 μM neutral lipid fluorescent probe BODIPY 493 / 503 diluted in PBS was added under light-protected conditions. Staining was performed for 30 minutes, followed by washing four times with PBS, and then 1 mL of NucBlue diluted in PBS was added. TM Nucleus fixation staining agent (2 drops of NucBlue per milliliter of PBS) TM Incubate with staining agent for 15 min, observe lipid droplet accumulation in cells under an inverted fluorescence microscope, and photograph representative areas. Determine intracellular triglyceride levels using a triglyceride kit, following the manufacturer's instructions and existing techniques.

[0051] S3: Isopropanol extraction and fluorescence intensity detection of intracellular BODIPY 493 / 503.

[0052] The instruments used in this step are: a full-wavelength multi-functional microplate reader, an inverted fluorescence microscope, a forced-air drying oven, a vortex mixer, and pipettes. Materials include: black 96-well plates, clear 96-well plates, 0.6 mL centrifuge tubes, a small shaker, pipette tips, and aluminum foil. Reagents include: PBS and isopropanol.

[0053] Aspirate the solution from the 6-well plate, allow it to dry, then add 300 μL of 100% isopropanol to each well. Wrap the wells in aluminum foil and place them on a shaker, shaking at low speed for 5 min. Transfer the solution from the plate to 0.6 mL EP centrifuge tubes using a pipette, dry the tubes in a 37°C oven, then reconstitute with 100 μL of isopropanol and vortex until homogeneous. Observe and photograph the fluorescence signal of residual cells in the plate after isopropanol treatment. Quantitatively transfer 45 μL of the isopropanol-reconstituted solution to a 96-well clear plate and measure the absorbance at 496 nm. Quantitatively transfer another 45 μL of the isopropanol-reconstituted solution to a 96-well black plate and measure the fluorescence intensity at 493 nm excitation / 514 nm emission wavelengths. Plot the results using Graphpad Prism 9 and analyze the experimental results. Compare the results with those obtained from commercially available kits to verify the reliability of the method.

[0054] Experimental results show that:

[0055] After treating HepG2 cells with 200 μM oleic acid (OA) for 24 h, the intensity of the green fluorescence signal in the cells was significantly higher than that in the DMSO control group, indicating that OA can promote triglyceride accumulation in HepG2 cells. After isopropanol extraction, the green fluorescence signal disappeared, but the blue fluorescence signal in the cell nucleus was not affected. Figure 3 By measuring the fluorescence intensity and absorbance of the isopropanol extract, the fluorescence signal intensity of the OA-treated group was found to be... Figure 4 A) and absorbance ( Figure 4 B) were significantly higher than the DMSO control group, and these results are consistent with those obtained from commercial triglyceride assay kits. Figure 4 C) indicates that isopropanol can be used to extract BODIPY 493 / 503 from stained cells, and the results of fluorescence intensity and absorbance detection reflect the changes in intracellular lipid content after compound treatment.

[0056] Example 2

[0057] This embodiment uses mouse C3H10T1 / 2 mesenchymal stem cells as the target cells and uses the Bodipy493 / 503 fluorescent probe to quantitatively analyze the intracellular lipid content. The specific steps are similar to those in Example 1. The main differences are explained below:

[0058] S2: C3H10T1 / 2 mesenchymal stem cell induction and rosiglitazone treatment

[0059] The instruments used in this step are: a full-wavelength multi-mode microplate reader, a carbon dioxide cell culture incubator, an inverted fluorescence microscope, a metal bath, a vortex mixer, and pipettes. Materials: C3H10T1 / 2 plates, 6-well plates, clear 96-well plates, 1.5mL centrifuge tubes, and pipette tips. Reagents: DMEM high-glucose medium (containing phenol red), penicillin / streptomycin, fetal bovine serum (Gibco), bone morphogenetic protein 7 (R&D Systems), indomethacin (Sigma), thyroid hormones (Sigma), 3-isobutyl-1-methylxanthine (Sigma), dexamethasone (Sigma), insulin (Sigma), Triton X-100 (Solepro), DMSO (Sigma), phosphate-buffered saline (PBS), isopropanol, Bodipy 493 / 503, and NucBlue. TM Invitrogen, a cell nucleus fixation staining agent TM Cell tissue fixative (Solepro), triglyceride assay kit (Pulley).

[0060] After resuscitation and passage culture, C3H10T1 / 2 mesenchymal stem cells were cultured at a rate of 2×10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well gelatin-coated plates. After cell attachment, the medium was replaced with medium containing 10 ng / μL bone morphogenetic protein 7 (BMP7) and cultured for another 3 days. When cells reached 100% confluence, differentiation was induced using an inducing agent consisting of 0.125 mM indomethacin, 1 nM thyroid hormone T3, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1 μg / mL insulin. Two days later, the medium was replaced with medium containing 1 μg / mL insulin and 1 nM T3 and cultured for another 6 days, changing the medium every 2 days. Brown adipocytes were obtained on day 8. From the start of differentiation induction, 2 μM rosiglitazone (Rosi) was added as the experimental group, and 0.1% DMSO (v / v) was used as the solvent control group. The treatment ended on day 8.

[0061] After exposure, the culture medium was discarded, and the cells were washed twice with PBS. 1 mL of cell fixative was added for fixation for 1 hour, followed by the addition of 1 mL of 0.1% Triton X-100 (v / v) to increase cell membrane permeability. After 15 minutes, 0.5 mL of 10 μM neutral lipid fluorescent probe BODIPY 493 / 503 diluted in PBS was added under light-protected conditions. Staining was performed for 30 minutes, followed by washing four times with PBS. Then, 1 mL of NucBlue diluted in PBS was added. TM Nucleus fixation staining agent (2 drops of NucBlue per milliliter of PBS) TMIncubate with staining agent for 15 min, observe lipid droplet accumulation in cells under an inverted fluorescence microscope, and photograph representative areas. Determine intracellular triglyceride levels using a triglyceride kit, following the manufacturer's instructions and existing techniques.

[0062] S3: Isopropanol extraction and fluorescence intensity detection of intracellular BODIPY 493 / 503.

[0063] The instruments used in this step are: a full-wavelength multi-functional microplate reader, an inverted fluorescence microscope, a forced-air drying oven, a vortex mixer, and pipettes. Materials include: black 96-well plates, clear 96-well plates, 0.6 mL centrifuge tubes, a small shaker, pipette tips, and aluminum foil. Reagents include: PBS and isopropanol.

[0064] Aspirate the solution from the 6-well plate, allow it to dry, then add 300 μL of 100% isopropanol to each well. Wrap the wells in aluminum foil and place them on a shaker, shaking at low speed for 5 min. Transfer the solution from the plate to 0.6 mL EP tubes using a pipette. Dry the centrifuge tubes in a 37°C oven, then add 100 μL of isopropanol to reconstitute the solution and vortex until homogeneous. Observe and photograph the fluorescence signal of the residual cells in the plate after isopropanol treatment. Quantitatively transfer 45 μL of the isopropanol reconstituted solution to a 96-well clear plate and measure the absorbance at 496 nm. Quantitatively transfer 45 μL of the isopropanol reconstituted solution to a 96-well black plate and measure the fluorescence intensity at 493 nm excitation / 514 nm emission wavelengths. Plot the results using Graphpad Prism 9 and analyze the experimental results. Compare the results with those obtained from commercially available kits to verify the reliability of the method of this invention.

[0065] Experimental results show that:

[0066] After treating C3H10T1 / 2 cells with 2 μM rosiglitazone (Rosi) for 8 days, the intensity of the green fluorescence signal in the cells was significantly higher than that in the DMSO control group, indicating that rosiglitazone can promote the differentiation and lipid accumulation of C3H10T1 / 2 cells. Figure 5 After staining, the green fluorescence signal disappeared after isopropanol extraction. Figure 5 By measuring the fluorescence intensity and absorbance of the isopropanol extract, the fluorescence signal intensity of the rosiglitazone-treated group was found to be... Figure 6 A) and absorbance ( Figure 6 B) were significantly higher than the DMSO control group, and these results are consistent with those obtained from commercial triglyceride assay kits. Figure 6 C) indicates that isopropanol can be used to extract BODIPY 493 / 503 from stained cells, and the results of fluorescence intensity and absorbance detection reflect the changes in intracellular lipid content after compound treatment.

[0067] In summary, the quantitative method of this invention is simple, fast, sensitive, effective, and has good experimental controllability and repeatability. The analytical results obtained are consistent with those obtained using other lipid content analysis methods, and it has important applications in the field of research on lipid metabolism interference effects of chemicals or environmental pollutants.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe, characterized in that, Includes the following steps: S1: Plotting fluorescence intensity on the ordinate and excitation or emission wavelength on the abscissa, curve fitting was performed to obtain the maximum excitation and emission wavelengths of the Bodipy 493 / 503 fluorescent probe in isopropanol solution at different concentrations. S2: After plating and culturing the target cells, they were divided into experimental and control groups. The experimental group was treated with compound exposure. Both the experimental and control groups were fixed with cell fixation solution and permeabilized with Triton X-100. Subsequently, Bodipy 493 / 503 fluorescent probes and NucBlue were used. TM Cellular staining agents were used to stain intracellular lipids and the cell nucleus, and finally observed and photographed under a fluorescence microscope; S3: Add 100% isopropanol to the cells of the control group and experimental group after treatment in step S2, shake and collect the extract. Observe the residual cells in the well plate under a fluorescence microscope and take pictures. Then, dry the extract and add 100% isopropanol to reconstitute it. Take the reconstituted solution and measure the fluorescence intensity and absorbance of the control group and experimental group reconstituted solutions under the maximum excitation wavelength and maximum emission wavelength, respectively. By comparing the fluorescence intensity and absorbance of the control group and experimental group reconstituted solutions, the quantitative analysis of lipid content in the target cells can be achieved.

2. The method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe according to claim 1, characterized in that, The specific steps of S1 are as follows: Bodipy 493 / 503 fluorescent probes were added to transparent 96-well plates, and the absorbance in the wavelength range of 400 nm to 700 nm was measured. The maximum absorption wavelength was obtained by curve fitting. Bodipy 493 / 503 fluorescent probes were added to 96-well black plates, and the emission wavelength was fixed at 702 nm. The fluorescence intensity after excitation by incident light in the wavelength range of 400 nm to 700 nm was scanned. Bodipy 493 / 503 fluorescent probes were added to 96-well black plates, and the excitation wavelength was fixed at 400 nm. The fluorescence intensity in the wavelength range of 402 nm to 700 nm was scanned. Then, with the fluorescence intensity as the ordinate and the excitation wavelength or emission wavelength as the abscissa, curve fitting was performed to obtain the maximum excitation wavelength and maximum emission wavelength of Bodipy 493 / 503 fluorescent probes in isopropanol solution at different concentrations.

3. The method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe according to claim 1, characterized in that, In step S1, the concentrations of the Bodipy 493 / 503 fluorescent probes are 0, 1, 2, 5, 10, 25, and 50 μM, respectively.

4. The method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe according to claim 1, characterized in that, In step S1, the maximum absorption wavelength, maximum excitation wavelength, and maximum emission wavelength of the Bodipy 493 / 503 fluorescent probe in isopropanol solution are 496 nm, 493 nm, and 514 nm, respectively.

5. The method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe according to claim 1, characterized in that, Step S2 is performed in a 6-well plate.

6. The method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe according to claim 1, characterized in that, In step S2, if the target cells are HepG2 cells or HK-2 cells, the compound exposure is achieved by treatment with 200 μM oleic acid; if the target cells are 3T3-L1 or C3H10T1 / 2 cells, the compound exposure is achieved by adding 2 μM rosiglitazone at the beginning of cell differentiation.

7. The method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe according to claim 1, characterized in that, In step S2, the concentration of the Bodipy 493 / 503 fluorescent probe is 10 μM, the volume is 0.5 mL, and the staining time is 30 min; NucBlue... TM The nuclear staining agent was prepared by diluting with PBS buffer, and 2 drops of raw NucBlue were added to each milliliter of PBS buffer. TM The staining agent was used, and the staining time was 15 minutes. All staining operations were performed under light-protected conditions.

8. The method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe according to claim 1, characterized in that, In step S3, the volume of isopropanol added for the first time is 0.3 mL, and the extraction time is 5 min; the volume of isopropanol added during reconstitution is 0.1 mL; the extraction and reconstitution operations are carried out under light-protected conditions.

9. The method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe according to claim 1, characterized in that, In step S3, the reconstituted solution is taken into a 96-well plate to measure the cell fluorescence intensity and absorbance.

10. The method for quantitative analysis of intracellular lipid content based on the Bodipy 493 / 503 fluorescent probe according to claim 1, characterized in that, In step S3, the volume of the reconstituted solution added is 45 μL, the absorbance detection wavelength is 496 nm, and the fluorescence detection excitation and emission wavelengths are 493 nm and 514 nm, respectively.

Images