A method for collecting and detecting lipid droplets

Abstract

This application relates to a method for collecting and detecting lipid droplets, comprising the steps of collecting lipid droplets by centrifugation, staining them with a neutral lipid dye, smearing the stained mixture, and observing it under a fluorescence microscope. This method for visually detecting lipid droplets is simple, feasible, and can intuitively record the size and number of lipid droplets.

Description

Technical Field

[0001] The present invention relates to the technical field of lipid droplets, and in particular to a method for collecting and detecting lipid droplets. Background Art

[0002] Lipid droplets (LDs) are spherical organelles found in nearly all cell types. Unlike other organelles, LDs are surfaced with a single phospholipid membrane, and their size changes dynamically in response to environmental influences. In mammalian cells, the primary component of the lipid droplet monolayer is phosphatidylcholine (PC), accounting for up to 60%, followed by phosphatidylcholine and phosphatidylinositol. Thousands of different structural and functional proteins, such as DGAT1, perilipin, and RAB proteins, are localized on the surface of LDs, regulating LD homeostasis and intracellular interactions. The core of the lipid droplet is composed of neutral lipids, such as triacylglycerols (TAGs) and sterol esters (SEs).

[0003] It is currently believed that intracellular lipid droplets serve as a lipid storage site, preventing excessive fatty acid accumulation within the cell and compromising membrane integrity. Furthermore, lipid droplets are important energy turnover sites, rapidly providing energy to the cell through interaction with mitochondria. As research deepens, it is discovered that intracellular lipid droplets have a wider range of functions than previously thought. For example, they participate in regulating the activation of proteins and signaling lipids in cells, serve as hubs for fatty acid transport, and are used as assembly platforms by viruses (Welte MA. Expanding roles for lipid droplets. Curr Biol. 2015 Jun 1). Increasing evidence indicates that intracellular lipid droplets are more than just lipid storage sites, especially for immune cells, whose activation is tightly regulated.

[0004] The function of lipid droplets in immune cells remains largely unclear, largely due to limitations in precise lipid droplet detection methods. While intracellular lipid droplets can typically be precisely measured using probes or fluorescent markers, there are currently no suitable methods for detecting lipid droplets in solution. Therefore, a simple and direct method to measure lipid droplet content in different sample types is urgently needed. Summary of the Invention

[0005] Through previous research, the inventors discovered that immune cells can accumulate a large number of lipid droplets under stress, and some of these droplets can be secreted by the cells. The secreted lipid droplets can also act as a medium for intercellular signaling, participating in cell activation. To this end, the present application provides a new method for detecting extracellular lipid droplets in immune cells, which can better visualize and observe the lipid droplets secreted by cells.

[0006] In one aspect, the present invention provides a method for collecting and detecting lipid droplets, the method comprising the following steps:

[0007] S1: Collecting lipid droplets: Centrifuging the sample containing lipid droplets to obtain cell culture supernatant containing lipid droplets;

[0008] S2: staining: adding a neutral lipid dye to the cell culture supernatant obtained in S1 to obtain a staining mixture;

[0009] S3: Smear and observation: The stained lipid droplets are encapsulated on a glass slide by the smear method and photographed and observed under a fluorescence microscope.

[0010] In a specific embodiment, in step S1, the lipid droplet-containing sample to be tested is any form of lipid droplet-containing sample, for example, it can be selected from in vitro cultured lipid droplet-containing cell samples (such as type 2 innate lymphocytes), tissue infiltrates (such as peritoneal lavage fluid and alveolar lavage fluid) and serum. Preferably, the in vitro cultured cell-containing sample is an in vitro cultured immune cell-containing sample.

[0011] In a specific embodiment, for the cell sample, the step S1 further comprises filtering the cell culture medium with a 0.22 μm filter.

[0012] In a specific embodiment, before step S1, 100-500 μl of culture medium is used to resuspend 4 -10 5 The cells were seeded into a cell culture plate and cultured in an incubator environment (5% CO2, 37°C) for 1-3 days.

[0013] In a specific embodiment, step S1 is further performed by the following steps:

[0014] S1-1: centrifuging the lipid droplet-containing sample at 300-500 g for 5-10 minutes at 4-16°C to collect the cell culture supernatant 1; and

[0015] S1-2: The cell culture supernatant 1 collected in step S1-1 is centrifuged at 3000-5000 g and 4-16° C. for 10-30 minutes to remove cell debris, and the cell culture supernatant 2 is collected.

[0016] In a specific embodiment, when the sample to be tested is tissue fluid, serum, and alveolar lavage fluid, after step S1-2, step S1-3 is further included:

[0017] The cell culture supernatant 2 is further purified by sucrose density gradient centrifugation to obtain cell culture supernatant 3. Step S1-3 is used to remove excess impurities in the solution to obtain purer lipid droplets.

[0018] In a specific embodiment, step S1-3 includes: mixing the cell culture supernatant 2 collected in step S1-2 and a 60wt% sucrose solution to form a mixed solution with a final sucrose concentration of 20wt%; adding 5wt% sucrose solution to the centrifuge tube through a sampling needle, then inserting the sampling needle into the bottom of the centrifuge tube and slowly adding the above-mentioned mixed solution with a final sucrose concentration of 20wt%, and finally covering the top layer of the gradient with a pH 7.4 PBS buffer, wherein the volume ratio of the 5wt% sucrose solution, the above-mentioned mixed solution with a final sucrose concentration of 20wt%, and the PBS buffer is 5:5:1; centrifuging at a speed of 20000-200000g for 30-120 minutes, and finally collecting the top layer of the solution accounting for 10vol% of the centrifuge tube as the cell culture supernatant 3.

[0019] In a specific embodiment, in step S2, the neutral lipid dye is selected from BODIPY 493 / 503, HCSLipidTOX TM .

[0020] In a specific embodiment, in step S2, the final concentration of the neutral lipid dye is 10 μM.

[0021] In a specific embodiment, in step S2, a neutral lipid dye is added to the cell culture supernatant obtained in S1 to obtain a staining mixture, which is vortexed and stained at room temperature for 5-10 minutes.

[0022] In a specific embodiment, in step S3, the smear method includes: dropping the dye mixture in the form of droplets into the middle of a clean glass slide, using a clean cover glass to align one end with the glass slide at a 30° angle, and slowly lowering the other end of the cover glass, and after the cover glass contacts the dye mixture in the middle of the glass slide, allowing it to naturally and slowly adhere, and after the cover glass and the glass slide are adhered, using a sealing agent to seal the gap between the cover glass and the glass slide.

[0023] In a specific embodiment, in step S3, the lipid droplet morphology is recorded using a Zeiss LSM900 laser confocal microscope 63X (NA1.4) objective lens under bright field and 488 nm laser, and the lipid droplet content is calculated using the following formula:

[0024] Lipid droplet content = number of lipid droplets / initial liquid volume.

[0025] Beneficial effects

[0026] The present invention provides a novel lipid droplet collection and detection method that can rapidly detect intact extracellular lipid droplets, is biotoxic and non-toxic, and maintains the biological activity of lipid droplets. Furthermore, the method provided herein is a visual detection method that can intuitively record the size and number of lipid droplets, providing a practical and feasible method for lipid droplet observation and research.

[0027] This application is the first to detect the presence of lipid droplets outside cells. Previously, there was no clear report that this micro-organelle structure could be secreted outside the cell. This application cleverly borrowed the method of cell counting to achieve the observation and counting of lipid droplets under a high-power microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 2: Fluorescence microscopy results of lipid droplets (collected in Example 1). In the figure, a represents the fluorescence signal intensity of BODIPY 493 / 503 under 488 nm laser conditions, b represents the results under bright field conditions, and c represents the superposition of the fluorescence signal and bright field conditions. The scale bar is 2 μm.

[0029] Figure 2 Schematic diagram of sucrose density gradient centrifugation. First, add 5 ml of a 5 wt% sucrose solution to the centrifuge tube via a pipette. Next, insert the pipette at the bottom and slowly add a 20 wt% sucrose solution mixed with the test solution. Finally, overlay the top layer of the gradient with 1 ml of pH 7.4 PBS buffer. Centrifuge at 180,000 g for 90 minutes.

[0030] Figure 3 Fluorescence microscopy results of lipid droplets (collected as in Example 2, without sucrose density gradient centrifugation). (a) shows the fluorescence signal intensity of BODIPY 493 / 503 measured under a 488 nm laser, (b) shows the intensity under bright field conditions, and (c) shows the superposition of the fluorescence signal and bright field conditions. The scale bar is 2 μm.

[0031] Figure 4 Fluorescence microscopy results of lipid droplets (collected in Example 2 and subjected to sucrose density gradient centrifugation). (a) shows the fluorescence signal intensity of BODIPY 493 / 503 detected under a 488 nm laser, (b) shows the result under bright field conditions, and (c) shows the superposition of the fluorescence signal and bright field conditions. The scale bar is 2 μm. DETAILED DESCRIPTION

[0032] The following describes in detail the lipid droplet collection and detection method of the present application through specific examples to enable those skilled in the art to better understand the content of the present application. However, these examples are not intended to limit the scope of the present application.

[0033] In this application, the numbers following “cell culture supernatant”, such as 1, 2 and 3, are only used to distinguish one cell culture supernatant from another, and have no other meanings.

[0034] Animals and cells: The mice used in the experiment were of C57BL / 6 background, and the cells were type 2 innate lymphoid cells from mouse lung tissue obtained by flow cytometry sorting.

[0035] Reagents: IL-2 (Biolegend, Cat#575406); IL-7 (Biolegend, Cat#577806); IL-33 (Biolegend, Cat#580506); BODIPY 493 / 503 (Thermo, Cat#D3922)

[0036] Instruments: Laser confocal microscope (Zeiss LSM900); Ultracentrifuge (Beckman XPN-100)

[0037] Example 1: Collection of lipid droplets from cells cultured in vitro

[0038] 1.10 4 Type 2 innate lymphocytes were reselected in 100 μl of culture medium (10% fetal bovine serum and 1% double-antibody) and stimulated with cytokines IL-2 (20 ng / ml), IL-7 (20 ng / ml), and IL-33 (20 ng / ml) for 3 days.

[0039] 2. Centrifuge at 500g for 5 minutes at 4°C and collect the cell culture supernatant into another 1.5ml centrifuge tube; then centrifuge at 3000g for 10 minutes at 4°C and collect the supernatant for later use.

[0040] 3. Neutral lipid dye BODIPY 493 / 503 (10 μM) was added to the supernatant to obtain a staining mixture, which was vortexed and stained at room temperature for 5 minutes.

[0041] 4. Take 20μl of the staining mixture and drop it in the middle of a clean slide. Use a clean cover glass to align one end with the slide at a 30° angle and slowly lower the other end of the cover glass. After the cover glass contacts the staining mixture in the middle of the slide, let it naturally and slowly fit together. Wait for 5 minutes for the cover glass and slide to fit together, and then use a sealing agent to seal the gap between the cover glass and the slide.

[0042] 5. Use Zeiss LSM900 laser confocal microscope 63X (NA1.4) objective lens to record lipid droplet morphology under bright field and 488nm laser (such as Figure 1 Finally, count all the lipid droplets under the microscope (for example, 200), and the lipid droplet content in the original solution is 10 / μl (200 / 20μl).

[0043] Example 2: Collection of lipid droplets from alveolar lavage fluid

[0044] 1. Induce the mouse asthma model by intranasal administration of IL-33 (500 ng) for 3 consecutive days, and collect the alveolar lavage fluid of the mice on the fourth day.

[0045] 2. Centrifuge at 500g for 5 minutes at 4°C and collect the supernatant of the lavage fluid into another 15ml centrifuge tube; then centrifuge at 3000g for 10 minutes at 4°C and collect the supernatant. At this step, reserve 100μl for direct staining observation. The results are as follows: Figure 3 As shown (staining and observation follow points 5-7 below).

[0046] 3. Take 4 ml of the supernatant and mix it with 2 ml of 60 wt% sucrose solution to make a mixed solution with a final sucrose concentration of 20 wt% for later use.

[0047] 4. First, add 5 ml of 5 wt% sucrose solution to the centrifuge tube through the injection needle. Then, insert the injection needle at the bottom and slowly add 5 ml of 20% mixed solution. Finally, cover the top layer of the gradient with 1 ml of PBS buffer (NaCl 8 g / L, Na2HPO4 1.42 g / L, KCl 0.2 g / L, KH2PO4 0.27 g / L, pH = 7.4). Centrifuge at 180,000 g for 90 minutes (as shown in the figure). Figure 2 ).

[0048] 5. Carefully collect 1 ml of the top layer of solution for subsequent staining, add neutral lipid dye BODIPY 493 / 503 (10 μM) to obtain a staining mixture, vortex, and stain at room temperature for 5 minutes.

[0049] 6. Take 20μl of the staining mixture and drop it in the middle of a clean slide. Use a clean cover glass to align one end with the slide at a 30° angle and slowly lower the other end of the cover glass. After the cover glass contacts the staining mixture in the middle of the slide, let it naturally and slowly fit together. Wait for 5 minutes for the cover glass and slide to fit together, and then use a sealing agent to seal the gap between the cover glass and the slide.

[0050] 7. Use Zeiss LSM900 laser confocal microscope 63X (NA1.4) objective lens to record lipid droplet morphology under bright field and 488nm laser (such as Figure 4 ).

[0051] By comparison, we can see that for alveolar lavage fluid samples, samples that have not been purified by sucrose density gradient centrifugation have a lot of impurities that interfere with the correct signal when observed under a microscope, which is easy to produce false positives ( Figure 3 ), after purification by sucrose density gradient centrifugation, the impurity signal observed under the microscope can be significantly reduced ( Figure 4 ), therefore, for non-in vitro cultured samples such as tissue fluid, serum, and alveolar lavage fluid, it is necessary to perform sucrose density gradient centrifugation to eliminate the interference of impurities in the solution.

Claims

1. A method for collecting and detecting extracellular lipid droplets of immune cells, the method comprising the following steps: S1: Collect lipid droplets: Centrifuge the sample containing lipid droplets to obtain the cell culture supernatant containing lipid droplets. Wherein, in step S1, the sample to be tested containing lipid droplets is selected from: Type 2 innate lymphocytes were stimulated with 20 ng / ml IL-2, 20 ng / ml IL-7, and 20 ng / ml IL-33 for 3 days, or The mouse asthma model was established by inducing IL-33 with 500 ng for 3 days, and the alveolar lavage fluid was collected on the fourth day; S2: staining: adding a neutral lipid dye to the cell culture supernatant obtained in S1 to obtain a staining mixture; S3: Smear and observation: The stained lipid droplets are encapsulated on a glass slide by the smear method and photographed and observed under a fluorescence microscope.

2. The method according to claim 1, wherein The step S1 is performed by the following steps: S1-1: Centrifuge the sample containing lipid droplets at 300-500 g for 5-10 minutes at 4-16°C, and collect the cell culture supernatant 1; as well as S1-2: The cell culture supernatant 1 collected in step S1-1 is centrifuged at 3000-5000 g and 4-16° C. for 10-30 minutes to remove cell debris, and the cell culture supernatant 2 is collected.

3. The method according to claim 2, wherein: When the sample to be tested is alveolar lavage fluid, after step S1-2, step S1-3 is further included: The cell culture supernatant 2 was further purified by sucrose density gradient centrifugation to obtain cell culture supernatant 3.

4. The method according to claim 3, wherein Step S1-3 includes: mixing the cell culture supernatant 2 collected in step S1-2 and a 60wt% sucrose solution to form a mixed solution with a final sucrose concentration of 20wt%; adding 5wt% sucrose solution to a centrifuge tube through a sampling needle, then inserting the sampling needle into the bottom of the centrifuge tube and slowly adding the mixed solution with a final sucrose concentration of 20wt%, and finally covering the top layer of the gradient with a pH 7.4 PBS buffer, wherein the volume ratio of the 5wt% sucrose solution, the mixed solution with a final sucrose concentration of 20wt%, and the PBS buffer is 5:5:1; centrifuging at a speed of 20000-200000g for 30-120 minutes, and finally collecting the top layer of the solution accounting for 10vol% of the centrifuge tube as the cell culture supernatant 3.

5. The method according to claim 1, wherein In step S2, The neutral lipid dye is selected from BODIPY 493 / 503, HCS LipidTOX™, and / or The final concentration of the neutral lipid dye was 10 µM.

6. The method according to claim 1, wherein In step S2, a neutral lipid dye is added to the cell culture supernatant obtained in S1 to obtain a staining mixture, which is vortexed and stained at room temperature for 5-10 minutes.

7. The method according to claim 1, wherein In step S3, the smearing method includes: dropping the dye mixture in the form of drops to the middle of a clean glass slide, using a clean cover glass to align one end with the glass slide at a 30° angle, and slowly lowering the other end of the cover glass. After the cover glass contacts the dye mixture in the middle of the glass slide, it is allowed to naturally and slowly adhere to the glass slide. After the cover glass and the glass slide are adhered, a sealing agent is used to seal the gap between the cover glass and the glass slide.

8. The method according to claim 1, wherein In step S3, lipid droplet morphology was recorded using a Zeiss LSM900 laser confocal microscope with a 63X objective under bright field and 488 nm laser, and the lipid droplet content was calculated using the following formula: lipid droplet content = number of lipid droplets / starting liquid volume.

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