Device and method for analyzing synaptic heterogeneity based on multicolor flow and application of device and method

Abstract

The invention provides a multicolor flow-based synaptic heterogeneity analysis device and analysis method and application thereof, the analysis device comprises: (1) a scattering-resolution pretreatment module: adopting a BD FACS fusion double laser platform, firstly positioning an FSC-SSC coordinate by using a standard 0.5 m polystyrene microsphere, and then loading a synaptic body; the adopted sheath fluid comprises HPMC (Hydroxymethyl Methacrylate)-3cp and 1 * PBS (Phosphate Buffer Solution) without Ca < 2 + > / Mg < 2 + >; an adopted carrier buffer solution comprises EDTA (Ethylene Diamine Tetraacetic Acid), a 1 * protease inhibitor mixture and 1 * PBS (Phosphate Buffer Solution) without Ca < 2 + > / Mg < 2 + >; and (2) a multi-color flow type synchronous marking-sorting module which is used for detecting two kinds of code fluorescent marks in parallel on double laser platforms of 488 nm and 561 nm, eliminating cross color by a logic gate-double exponential decay compensation algorithm, and realizing transmitter and function sorting.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to a synaptic heterogeneity analysis device and analysis method based on multicolor flow cytometry and its application. Background Technology

[0002] The brain is a highly complex neural network system whose function depends on the transmission of information between neurons via synapses. Synapses are not only the basic structural units for communication between neurons, but also exhibit significant heterogeneity in type, function, and molecular composition. Based on different neurotransmitter types, synapses can be classified into various subtypes, such as glutamatergic synapses, GABAergic synapses, and dopaminergic synapses; furthermore, synapses can be further subdivided according to their functional states (such as activation state and plasticity level). Revealing the molecular composition of different types of synapses is of great significance for understanding the functional regulation mechanisms of neural networks and the pathogenesis of neurological diseases.

[0003] However, due to the high complexity of the structure and composition of synaptosomes, traditional biochemical separation methods struggle to achieve efficient and precise separation of specific types of synaptosomes. In recent years, flow cytometry, with its high throughput and quantitative analysis capabilities, has been increasingly applied to synaptosome research, becoming an emerging synapse sorting technology.

[0004] Currently, mainstream synaptic flow cytometry sorting techniques primarily rely on monochromatic flow cytometry, which uses genetic engineering to express fluorescent protein markers in specific types of synapses. For example, researchers can use transgenic mouse models to specifically express green fluorescent proteins (such as tdTomato) in glutamatergic neurons, thereby enabling their synaptosomes to carry fluorescent signals. Subsequently, flow cytometry is used to detect the fluorescence intensity of each synaptosome, and sorting is performed based on the intensity of the fluorescence signal. By performing mass spectrometry analysis on synaptosomes carrying different fluorescent signals, their differences in protein composition can be systematically resolved, thus revealing the molecular basis of synaptosome heterogeneity.

[0005] Proximity labeling and fluorescence-activated synaptosome sorting are cutting-edge techniques for the separation of subcellular structures. Proximity labeling uses gene editing to anchor enzymatic tags (such as APEX and BioID) to known synaptic proteins, marking spatially neighboring proteins through enzymatic catalysis. The labeled proteins are then identified by mass spectrometry, allowing for the inference of the synaptic complex composition. However, this technique has the following inherent limitations in synapse research: (1) Insufficient spatial specificity: Synaptic proteins are mainly synthesized in the cell body and transported to the synapse along the axon. During the transport process, the enzymatic tags may diffuse to non-synaptic regions, resulting in a large number of mislabeled proteins in the cell body, axon membrane, or myelin, leading to high background signal. (2) Dependence on prior targets: Synaptic proteins that are known and have antibody or gene editing capabilities must be used as anchoring sites. Research on synaptic subpopulations with unknown functions or for which no markers have been found cannot be carried out, limiting the discovery of new synaptic types.

[0006] Fluorescence-activated synaptosome sorting is currently the mainstream strategy for isolating neurotransmitter-specific synapses: specific neurotransmitter synapses are genetically modified to express fluorescent proteins, and synapses are extracted using a mechanochemical method and then sorted by fluorescence intensity using flow cytometry. However, FM membrane dyes limit multicolor schemes: synapse diameters are only ~0.5-1 µm, overlapping with cell debris and lipid droplet sizes, requiring broad-spectrum FM membrane dyes to set forward scattering (FSC) / side scattering (SSC) thresholds to exclude non-synaptic particles. FM dyes have broad emission spectra (500-700 nm), resulting in severe color cross-contamination with conventional fluorescent proteins (GFP, mCherry, BV421, etc.), meaning traditional schemes can only use a single fluorescence channel and cannot simultaneously resolve neurotransmitter type and functional state. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a multicolor flow cytometry-based synaptic heterogeneity analysis device and method, as well as their applications. The analysis device and method of the present invention achieve two-dimensional sorting based on "neurotransmitter type × functional state," and combined with subsequent mass spectrometry analysis, simultaneously generate proteomic maps of multiple heterogeneous synapses, providing a general technical platform for discovering novel synaptic subpopulations and elucidating their molecular mechanisms under physiological and pathological conditions.

[0008] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a synaptic heterogeneity analysis device based on multicolor flow cytometry, the analysis device comprising: (1) Scattering-resolution pretreatment module: The BD FACS fusion dual-laser platform was used. First, the FSC-SSC coordinates were located using standard 0.5 µm polystyrene microspheres, and then the synaptic bodies were loaded. The sheath fluid used consisted of HPMC-3cp and 1×Ca-free solution. 2+ / Mg 2+ PBS, wherein the concentration of HPMC-3cp is 0.02-0.08% w / v; the sheath fluid has a viscosity of 1.6-2.0 cP and an ionic strength of 160-220 mOsm kg. -1 The pH was 7.35-7.45; the carrier buffer used consisted of EDTA, 1× protease inhibitor mixture, and 1× Ca2+-free buffer.2+ / Mg 2+ PBS, wherein the concentration of EDTA is 18-20 mM; the ionic strength of the carrier buffer is 160-220 mOsm kg. -1 Viscosity 1.6-2.0 cP, pH 7.35-7.45; (2) Multicolor streaming synchronous marking-sorting module: On the dual laser platform of 488 nm and 561 nm, two kinds of fluorescent markers are detected in parallel, and cross-coloring is eliminated by logic gate-double exponential decay compensation algorithm to achieve quality and function sorting.

[0009] In this invention, the concentration of HPMC-3cp in the sheath fluid is 0.02-0.08% w / v, for example, 0.02% w / v, 0.04% w / v, 0.06% w / v, or 0.08% w / v; the viscosity of the sheath fluid is 1.6-2.0 cP, for example, 1.6 cP, 1.8 cP, or 2.0 cP; and the ionic strength is 160-220 mOsm kg. -1 For example, it could be 160 mOsm kg -1 180 mOsmkg -1 200 mOsm kg -1 Or 220 mOsm kg -1 The pH value is 7.35-7.45, for example, it can be 7.35, 7.4 or 7.45, etc.

[0010] In this invention, the concentration of EDTA in the carrier buffer is 18-20 mM, for example, 18 mM, 19 mM or 20 mM; the viscosity is 1.6-2.0 cP, for example, 1.6 cP, 1.8 cP or 2.0 cP; and the pH is 7.35-7.45, for example, 7.35, 7.4 or 7.45.

[0011] This invention addresses the "monochromatic" bottleneck in existing technologies by proposing a multicolor flow cytometry synaptosome sorting strategy that is independent of FM dyes. By optimizing the sheath fluid ionic strength, viscosity, and the osmotic pressure, pH, and surfactant combination of the carrier solution, the scattering resolution of synaptosome particles in flow cytometry detection is significantly improved, allowing them to be distinguished from debris noise without membrane dyes. After eliminating the spectral occupancy of FM dyes, ≥2 fluorescent labels (such as neurotransmitter markers - mCherry, activity markers - pHluorin, and plasticity markers - BV421) can be used in parallel to achieve two-dimensional sorting based on "neurotransmitter type × functional state." Combined with subsequent mass spectrometry analysis, proteomic maps of multiple heterogeneous synapses can be constructed simultaneously, providing a general technical platform for discovering novel synaptic subpopulations and elucidating their molecular mechanisms under physiological and pathological conditions.

[0012] Preferably, the protease inhibitor mixture comprises: AEBSF, Aprotinin, Leupeptin, Bestatin, E-64, and Pepstatin A.

[0013] In this invention, the protease inhibitor mixture used is: protease inhibitor Cocktail, EDTA-free, 100×DMSO stock solution, from Yisheng Company, catalog number 20124ES03.

[0014] Preferably, the 1×Ca-free 2+ / Mg 2+ PBS was prepared from ultrapure water with a resistivity ≥18 MΩ·cm.

[0015] Preferably, the sheath fluid is used after being filtered through a 0.22-0.25 µm filter membrane.

[0016] Preferably, the carrier buffer solution is used after being filtered through a 0.22-0.25 µm filter membrane.

[0017] In this invention, a specific ionic strength (180 mOsm kg) is used. -1 Sheath fluid with viscosity (1.8 cP, HPMC-3 cp 0.05% w / v) and pH 7.40 ± 0.05, and containing 20 mM EDTA and free of Ca 2+ / Mg 2+ The ultrapure PBS carrier buffer, in synergy with the 0.5-1 µm synaptosome, enables reproducible separation of electron / sheath fluid noise and cell debris in the FSC-SSC two-dimensional scatter plot (noise <2%, debris <10%, recovery rate >90%), thus completely eliminating the need for FM membrane dyes and their spectral occupancy.

[0018] Preferably, the fluorescent marker includes: a neurotransmitter type marker and a functional status marker; The neurotransmitter type marker is the synaptophysin-tdTomato fusion protein, with an excitation wavelength of 561 nm and an emission wavelength of 610 / 20 nm; the functional state marker is FM4-64, with an excitation wavelength of 488 nm and an emission wavelength of 620 / 30 nm.

[0019] Preferably, the logic gate-double exponential decay compensation algorithm includes: (a) Single-stain microsphere calibration: Thermo UltraRainbow 0.5 µm microspheres were used to single stain two types of fluorescence, 10,000 events were collected, and a 2×2 spectral overlap matrix was established; (b) Double exponential decay compensation algorithm: Let I raw I represents the original fluorescence intensity. corr To compensate for the strength, then, ; Parameters k1-k4 were obtained by Levenberg-Marquardt nonlinear fitting, and the residual crosstalk after compensation was <5%. (c) Logic gate sorting: P1: FSC-SSC elliptical gate, delineating synaptosome groups; P2: vGlut1 / GAD2 / DAT×AI34, neurotransmitter gating, threshold set to mean + 3 SD for single-staining positive microspheres; P3 / P4: FM4-64, a functional gate with threshold values ​​at the 20% of strongest and weakest fluorescence intensities; Sorting P3 / P4: P3: vGlut1 + / FM4-64 + Activates glutamatergic energy; P4: vGlut1 + / FM4-64 - Resting glutamate can.

[0020] In this invention, the sorting parameters are set as follows: nozzle: 70 µm; sheath fluid pressure: 4.5 psi; drop-drive frequency: 30 kHz; sorting mode: 2-way purity; event rate: 18000-24000 s. -1 .

[0021] In this invention, ≥2 fluorescent markers (neurotransmitter type markers such as synaptophysin-tdTomato, and functional / plasticity markers such as FM4-64 or pHluorin) are simultaneously detected under a 488 nm + 561 nm dual laser platform. A "double exponential decay" nonlinear compensation algorithm (residual crosstalk <5%) is used to achieve two-dimensional logic gate sorting of "neurotransmitter type × functional state" for the first time, resulting in ≥4 subgroups such as activating / resting glutamatergic, GABAergic, and dopaminergic.

[0022] Secondly, this invention provides a method for analyzing synaptic heterogeneity based on multicolor flow cytometry, the method comprising: (1) Collect synaptosome samples from brain tissue; (2) Using the BD FACS fusion dual-laser platform, the FSC-SSC coordinates were first located with standard 0.5 µm polystyrene microspheres, and then the collected synaptic body samples were loaded. (3) FSC-SSC first sets up a gate to exclude noise and debris; (4) Dual-channel acquisition at 488 nm / 561 nm with real-time compensation; (5) The logic gate is divided into two paths, and the two collection tubes are connected to P3 / P4 respectively.

[0023] In this invention, after sorting, synaptosomes can be subsequently verified by Western blot, mass spectrometry, or electrophysiology.

[0024] In step (1) of this invention, the step of collecting synaptosome samples from brain tissue includes: homogenizing fresh brain tissue in sucrose-HEPES buffer containing protease inhibitors at 4°C; centrifuging at 1000 g for 10 min at 4°C to remove the nucleus; and transferring the supernatant to a solution containing 1.2 mol / L... -1 Centrifuge the top of the sucrose in HEPES buffer at 106000 g for 1 h at 4 °C; collect 0.8 / 1.2 mol L. -1 The sucrose interface band was resuspended in carrier buffer and filtered at 0.22 µm for later use.

[0025] The sucrose-HEPES buffer containing the protease inhibitor comprises: 0.32 mol L -1 Sucrose, 4 mmol / L -1 HEPES-NaOH, pH 7.4.

[0026] Preferably, in step (4), the parameters of the real-time compensation are set as follows: double exponential decay compensation, residual crosstalk <5%.

[0027] Preferably, in step (5), the parameters of the logic gate two-way sorting are set as follows: P1 elliptical gate FSC-SSC delineates the synaptosome; P2 neurotransmitter gate threshold is set to mean + 3 SD of single-stained positive microspheres; P3 / P4 functional gate thresholds are respectively set to 20% of the strongest and 20% of the weakest fluorescence intensity of FM4-64.

[0028] Thirdly, the present invention provides the application of the synaptic heterogeneity analysis device based on multicolor flow cytometry described in the first aspect or the synaptic heterogeneity analysis method based on multicolor flow cytometry described in the second aspect in synaptic detection.

[0029] In this invention, the above-mentioned multicolor flow cytometry-based synaptic heterogeneity analysis device and analysis method are used to draw a "neurotransmitter-function" two-dimensional synaptosome proteome map, which can be used to discover unknown synaptic subpopulations, screen disease-specific synaptic biomarkers or drug targets.

[0030] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0031] Compared with the prior art, the present invention has the following beneficial effects: (1) Dimensional increase: Existing technologies can only distinguish the type of neurotransmitter through a single fluorescence; the present invention introduces a second or even a third fluorescence simultaneously without the use of FM dye, realizing "neurotransmitter × function" two-dimensional sorting, increasing the information dimension by ≥2 times.

[0032] (2) Simultaneous improvement in purity and recovery rate: Traditional PBS sheath fluid fragmentation events are 16-20% and noise is more than 5%; the present invention reduces fragmentation to <10% and noise to <2%, while the synaptosome recovery rate remains >90%, significantly reducing the background of downstream mass spectrometry.

[0033] (3) Controllable spectral cross-color: For the first time, “double exponential decay” nonlinear compensation was applied to 0.5-1 µm particles, with residual cross-color <5%, which is much lower than the 15-20% of conventional linear compensation, reducing the false positive rate of weak positive population sorting by more than 3 times.

[0034] (4) Versatility and cost advantages: No transgenic FM dyes or expensive antibody-magnetic beads are required. The instrument can be upgraded on a conventional flow cytometer simply by adjusting the sheath fluid / carrier formulation. The reagent cost is reduced by about 60%, and the operation time is shortened by 1 / 3.

[0035] (5) Direct support function study: The integrity of the synaptosome after sorting (electrophysiological EPSC / mIPSC amplitude) is no different from that of the traditional method, but the subgroup resolution is improved, and the proteomic differences between the "activated-resting" states can be obtained in one experiment. Attached Figure Description

[0036] Figure 1 The results are for Comparative Example 1.

[0037] Figure 2 The results are from Example 1.

[0038] Figure 3 The results are from Example 2. Detailed Implementation

[0039] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0040] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0041] In this invention, a "sheath fluid-carrier dual-adjustment" strategy is employed to achieve electronic and sheath fluid noise levels below 2% and cell debris levels below 10% on a 0.5-1 µm synaptosome FSC-SSC two-dimensional scatter plot without the use of FM membrane dyes. Two types of fluorescent markers (including neurotransmitter type markers and functional state markers) are detected in parallel on a dual-laser platform at 488 nm and 561 nm, and cross-coloring is eliminated using a "logic gate-double exponential decay" compensation algorithm, achieving "neurotransmitter × function" sorting.

[0042] The technical objective of this invention is to enable 0.5-1 µm synaptosomes to simultaneously satisfy the following conditions on the FSC-SSC two-dimensional scatter plot without using FM (membrane dye): ① electronic noise + sheath fluid noise < 2% (based on the total number of events); ② cell debris events < 10%; ③ synaptosome recovery rate > 90%.

[0043] In verifying the technical effectiveness of this invention, a BD FACS fusion dual-laser platform was used. First, standard 0.5 µm polystyrene microspheres were used to locate the FSC-SSC coordinates. Then, mouse cortical synapses were loaded. Using the traditional PBS sheath fluid group, fragmentation events were 16.7%, and noise events were approximately 5%. Using the optimized "sheath fluid-carrier" method of this invention, fragmentation events were 9.2%, and noise events were approximately 1.6%, meeting the design requirements.

[0044] Example 1 This embodiment provides a method for analyzing synaptic heterogeneity based on multicolor flow cytometry.

[0045] 1. Laboratory animals and samples.

[0046] Forty mice (n=6 / group) aged 8-16 weeks were selected, consisting of wild-type C57BL / 6J mice and three transgenic mice: vGlut1-Cre::Ai34-tdTomato, GAD2-Cre::Ai34, and DAT-Cre::Ai34. Half were male and half female.

[0047] 2. Collect synaptosome samples.

[0048] (1) Brain tissue homogenate.

[0049] After cutting up a single cortex / hippocampus (approximately 100 mg), transfer it to a 2 mL Dounce glass homogenizer, add 1 mL of pre-cooled sucrose buffer, and homogenize at a slow speed of 10 cycles followed by a high speed of 10 cycles, all on ice.

[0050] (2) Sucrose gradient centrifugation.

[0051] The homogenate was incubated at 1000 g for 10 min at 4°C to remove the nuclei. The supernatant was added to the top of a three-layer sucrose gradient (0.8 M / 1.0 M / 1.2 M, 1 mL each) and incubated at 100,000 g for 1 h at 4°C (Beckman SW41Ti). The pale yellow band (rich in synaptosomes) at the 1.0 / 1.2 M interface was collected, diluted with 4-fold volume of sucrose buffer, and incubated at 20,000 g for 12 min at 4°C to precipitate the synaptosomes. Finally, the protein was resuspended in 200 µL SB, quantified with BCA, and the protein concentration was adjusted to 0.5 mg / mL. -1 ,spare.

[0052] 3. The BD FACS fusion dual-laser platform was used to detect synaptosome samples.

[0053] (1) Scattering-resolution preprocessing (Module A verification).

[0054] ① Instrument calibration: Use 0.5 µm polystyrene microspheres (Thermo) to set the FSC-SSC voltage to 280 / 400 V and the threshold FSC to 500; ② Sheath fluid replacement: Replace the original PBS sheath fluid with SF (180 mOsm kg). -1 (1.8 cP, 0.05% HPMC-3cp), system flush for 30 min; ③ Sample loading: Take 50 µL of synaptosome suspension (0.5 mg / mL) -1 Dilute to 500 µL SF, and control the event rate at 18000-24000 s. -1 ; ④ Set up a gate: an elliptical gate P1 delineates a region of 0.5-1 µm, records 9.1% of fragmentation events, 1.5% of noise, and 92% of synaptosome recovery rate (n=6).

[0055] (2) Multicolor fluorescent labeling.

[0056] ① Neurotransmitter markers: This embodiment uses transgenic endogenous tdTomato, which does not require external staining; ② Functional markers: Add FM4-64 to a final concentration of 5 µM, incubate at 37°C for 2 min, then immediately place on ice for 5 min to terminate staining; ③ Compensation microspheres: 10,000 events were set for each of the single-stained UltraRainbow 0.5 µm microspheres, and a 2×2 matrix was established. The double exponential decay algorithm (k1-k4 fitting) was used for compensation, and the residual cross-coloring was 4.2%.

[0057] (3) Logic gate sorting.

[0058] P1: FSC-SSC synaptosome main group; P2: tdTomato positive (561 nm, 610 / 20); P3: FM4-64 highly positive (488 nm, 620 / 30, Top 20%); P4: FM4-64 negative (Bottom 20%). Two-way sorting mode: P3 (activating glutamatergic) and P4 (resting glutamatergic), yield 87%, purity 90% (verified by secondary sorting after secondary processing).

[0059] Comparative Example 1 This comparative example provides a synaptic analysis method. The only difference between this method and Example 1 is that the sheath fluid used is PBS, while the remaining steps are the same as in Example 1.

[0060] In Example 1, fragmentation events were 9.2% ± 0.7%, noise was 1.6% ± 0.3%, and synaptosome recovery was 92% ± 3% (BCA protein quantification). In Comparative Example 1, fragmentation events were 16.7% ± 1.2%, and electron + sheath fluid noise was 5.1% ± 0.4%.

[0061] A comparison of the results from Example 1 and Comparative Example 1 shows that the low viscosity of PBS (1.0 cP) results in insufficient refractive index difference between 0.5-1 µm particles and the buffer solution, leading to decreased scattering resolution. Simultaneously, the lack of EDTA to inhibit metalloproteinases reduces synaptic membrane integrity and increases the fragmentation ratio. This invention, through a "sheath fluid-carrier dual-adjustment" strategy, can reduce background noise by 70% and increase recovery rate by 18% without the need for FM membrane dyes, thereby directly supporting the purity of subsequent multicolor sorting.

[0062] Figure 1 The results for Comparative Example 1 are shown in the figure. The scattering resolution is reduced, and the synaptosomes and adhesion bodies cannot be effectively distinguished.

[0063] Figure 2 The results are from Example 1. Figure 2 The synaptosomes and adhesion bodies are clearly separated, with the synaptosomes accounting for a larger proportion.

[0064] Example 2 This embodiment verifies the feasibility of multicolor sorting. Traditional synaptosome flow cytometry relies on membrane dyes with extremely broad emission spectra (such as FM1-43) to set fluorescence thresholds (typically >700 AU), combined with SSC>200 to eliminate basic noise. However, spectral crosstalk from membrane dyes severely limits multicolor labeling capabilities, enabling synaptosomes to be sorted only in a single color. To overcome this bottleneck, this embodiment, without using any membrane dye fluorescence thresholds, uses only SSC>200 as the sole detection threshold to verify whether the scattering-resolution system of this invention can independently achieve effective two-color sorting.

[0065] Fluorescent labeling combination: Neurotransmitter type: vGlut1-tdTomato (561 nm, 610 / 20).

[0066] Functional status: FM4-64 (488 nm, 620 / 30).

[0067] Compensation and Logic Gates: A 2×2 spectral overlap matrix was established by staining 0.5 µm microspheres with 10,000 events each; after compensation using a double exponential decay algorithm, the residual cross-coloring was 4.3% ± 0.6%.

[0068] Logic gate settings: P1: FSC-SSC Elliptical Gate; P2: tdTomato positive (mean + 3 SD); P3: High positivity for FM4-64 (Top 20%) P4: FM4-64 low positive (Bottom 20%).

[0069] Sorting results: In two-way sorting mode (70 µm nozzle, 4.5 psi, 30 kHz): vGlut1 + / FM4-64 + (Activated glutamate) Yield 42%, purity 90%±2%; vGlut1 + / FM4-64 - (Restless glutamic acid) yield 45%, purity 89%±3%; Total event rate 18000-24000s -1 After sorting, the samples were tested again to verify that there was no cross-contamination.

[0070] Conclusion: Under the premise of no FM membrane dye occupying the channel, dual laser and dual fluorescence can achieve two-dimensional sorting of "transmitter × function" with residual cross-coloring of less than 5%, which meets the downstream needs of high-throughput proteomics.

[0071] Figure 3 The test results are for Example 2. Figure 3 The scattering-resolution system of the present invention can achieve two-color flow cytometry without cross-contamination.

[0072] Example 3 This embodiment provides a method for analyzing synaptic heterogeneity based on multicolor flow cytometry. The only difference between this embodiment and Embodiment 1 is that the sheath fluid-carrier used is the official BD FACS™ sheath fluid (Cat. 342003, KCl concentration ≈ 25 mM, total ionic strength 180 mOsm kg). -1(Viscosity 1.0 cP, pH 7.0).

[0073] Experimental Design: The process remained completely identical to Example 1, except that the "homemade SF-SB system" was entirely replaced with the official BD FACS™ sheath solution (Cat. 342003, KCl concentration ≈ 25 mM, total ionic strength 180 mOsm kg). -1 (Viscosity 1.0 cP, pH 7.0). Remaining steps: Sucrose gradient purification → 0.5 mg mL -1 Resuspension → Add FM4-64 (5 µM, 37℃ for 2 min) → Ice bath for 5 min → BDFACSFusion dual-laser detection.

[0074] Key phenomena: (1) FM4-64 fluorescence decay curve: In BD sheath fluid, the average fluorescence intensity (MFI) of the 488 nm channel decreased by 42% within 10 min; while the SF-SB (K) of this invention... + (≤5 mM) No significant change.

[0075] (2) Spectral stability: The cross-color coefficients of the 561 nm / 488 nm channels in the BD group drifted over time. After 10 min, the compensation matrix deviation was >12%, which caused the P3 (FM4-64 high positive) gate boundary to be unable to be locked, and the sorting purity dropped from the initial 80% to 63%.

[0076] Mechanism Analysis: FM4-64 is a positively charged styrene dye whose membrane binding depends on the negatively charged phospholipids (PS / PI) in the outer leaflets. High concentrations of K... + (≥20 mM) By shielding surface charge and reducing film potential, the dye-film electrostatic interaction is weakened; at the same time, high K + Inducing synaptosome depolarization, activating vesicle cycling, and accelerating dye endocytosis and quenching in the acidic intracellular pool lead to rapid and irreversible decay of the "functional gate" signal. The SF-SB system will... + Controlled within ≤5 mM (Na) + Maintaining a film potential of -60 mV, significantly suppressing dye translocation, and ensuring stable signal in the 488 nm channel.

[0077] Conclusion: Although BD's official sheath solution meets the requirements for conventional cell scattering, its high KCl formulation (≥25 mM) directly disrupts the membrane-binding stability of FM4-64, causing functional state fluorescence gating to fail, crosstalk >12%, and a decrease in sorting purity >15%. This invention addresses this issue through a "low KCl" approach. +The SF-SB system, consisting of "≤5 mM + viscosity fine-tuning + EDTA chelation," not only reduces background noise and debris but, more importantly, maintains the positioning stability of the FM4-64 membrane, with fluorescence decay of <8% within 10 minutes, ensuring long-term reproducibility of the "neurotransmitter × function" two-dimensional gating. Therefore, if a commercial sheath solution is used, it must be additionally diluted to K. + To achieve the same scattering-resolution and multicolor stability as this invention, ≤5 mM EDTA plus 0.05% HPMC-3 cp and 20 mM EDTA is required.

[0078] Example 4 This embodiment provides a method for analyzing synaptic heterogeneity based on multicolor flow cytometry.

[0079] 1. Experimental objective.

[0080] We will verify whether stable tricolor synaptosome sorting can still be achieved under the dual laser platform of "488 nm + 561 nm" by replacing the functional state dye FM4-64 with QuantumDot (QD)-Streptavidin-antibody conjugate and introducing MitoTracker Green (MT488) as mitochondrial integrity gating.

[0081] 2. Fluorescent labeling combination.

[0082] Transmitter type: Ai34-tdTomato (vGlut1-Cre::Ai34, 561 nm excitation, 610 / 20 emission) – consistent with Example 1.

[0083] Functional status: QD705-Streptavidin + biotinylated anti-Synaptotagmin-1 antibody (488 nm excitation, 710 / 50 emission).

[0084] Mitochondrial status: MitoTracker Green FM (MT488, 488 nm excitation, 530 / 30 emission, 100 nM, 37℃ for 20 min followed by ice bath rinse).

[0085] 3. Experimental steps.

[0086] ① The synaptosomes purified by the sucrose gradient were first resuspended in SB + 20 mM EDTA, 0.5 mg / mL -1 .

[0087] ② First add biotinylated anti-Syt1 (1:200, 4℃ 30 min), wash once with PBS; then add QD705-Streptavidin (1:400, 4℃ 20 min, protected from light).

[0088] ③ Then add 100 nM MT488, incubate at 37°C for 20 min, and immediately wash twice with excess SB to remove free dye.

[0089] ④ Installation: BD FACSFusion, 70 µm nozzle, SF-SB sheath fluid, event rate <5000 s -1 The compensation matrix was constructed using single-stained UltraRainbow microspheres and MT488 single-stained microspheres, respectively.

[0090] 4. Key Results.

[0091] 4.1 QD705 marker stability.

[0092] Multiple independent replicates (n = 6) showed that the QD705-Syt1 labeling success rate was only 50% (3 / 6 batches).

[0093] Successful batches: Only 5% of synaptosomes were QD705 positive.

[0094] Failed batch: QD signal was almost undetectable in synaptosomes, and there was an increase in free bright spots in the background; the membrane colocalization rate under confocal microscopy was <5%, which was no different from the "unlabeled" control.

[0095] Reason: QD-Streptavidin conjugates are prone to reversible aggregation in the high ionic strength of 180 mOsm SF-SB, and the sulfated proteoglycans on the synaptosome membrane surface compete with QD for binding, resulting in highly unstable antibody-QD bridging efficiency between batches. In summary, QD lacks reproducible membrane anchoring ability and cannot meet the quantitative requirements of functional gating.

[0096] 4.2 Mitochondrial gating stability of MT488.

[0097] Within 30 minutes, the MFI of MT488 decreased by only 9%, and the CV was less than 5%.

[0098] The FSC-SSC-MT488 three-dimensional gating system can separate "containing mitochondrial synaptosomes" from "without mitochondrial fragments" with a recovery rate of 93% and a purity of 91%.

[0099] 4.3 Three-color flow cytometry representation.

[0100] Due to the continuous attenuation of the QD signal, the QD channel was ultimately abandoned as a function gate, and only the following was retained: P1: FSC-SSC Elliptical Gate; P2: Ai34-tdTomato positive (neurotransmitter); P3: High MT488 positivity (intact mitochondria).

[0101] This yields "vGlut1"+ / MT488 + "The population, with a purity of 90% and a yield of 88%, highly overlapped with the results of the FM4-64 functional gate in Example 1 (r=0.93, p<0.001).

[0102] 5. Conclusion.

[0103] The QD705-Streptavidin antibody system exhibits insufficient stability when labeled on 0.5-1 µm synaptosome surfaces, with a signal loss of >60% over 30 minutes, making it unsuitable for functional state gating.

[0104] MitoTracker Green FM can stably label residual mitochondria in the synapse, with a signal drift of <10% after 30 min. Together with Ai34-tdTomato and traditional FM4-64, it forms a new two-dimensional gating system for "neurotransmitter × mitochondrial integrity".

[0105] With a dual-laser platform, the "Ai34-tdTomato+MT488" can achieve high-purity three-color flow cytometry (tdTomato red, MT488 green, SSC trigger), which can meet the synaptic subpopulation sorting requirements without the need for an additional 640 nm laser, providing a closed process that can be directly docked for subsequent mitochondrial-synaptic coupled proteomics research.

[0106] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A synaptic heterogeneity analysis device based on multicolor flow cytometry, characterized in that, The analytical device includes: (1) Scattering-resolution pretreatment module: The BD FACS fusion dual-laser platform was used. First, the FSC-SSC coordinates were located using standard 0.5 µm polystyrene microspheres, and then the synaptic bodies were loaded. The sheath fluid used consisted of HPMC-3cp and 1×Ca-free solution. 2+ / Mg 2+ PBS, wherein the concentration of HPMC-3cp is 0.02-0.08% w / v; the sheath fluid has a viscosity of 1.6-2.0 cP and an ionic strength of 160-220 mOsm kg. -1 The pH was 7.35-7.45; the carrier buffer used consisted of EDTA, 1× protease inhibitor mixture, and 1× Ca2+-free buffer. 2+ / Mg 2+ PBS, wherein the concentration of EDTA is 18-20 mM; the ionic strength of the carrier buffer is 160-220 mOsm kg. -1 Viscosity 1.6-2.0 cP, pH 7.35-7.45; (2) Multicolor streaming synchronous marking-sorting module: On the dual laser platform of 488 nm and 561 nm, two kinds of fluorescent markers are detected in parallel, and cross-coloring is eliminated by logic gate-double exponential decay compensation algorithm to achieve quality and function sorting.

2. The synaptic heterogeneity analysis device based on multicolor flow cytometry according to claim 1, characterized in that, The mixture of protease inhibitors includes: AEBSF, Aprotinin, Leupeptin, Bestatin, E-64, and Pepstatin A.

3. The synaptic heterogeneity analysis device based on multicolor flow cytometry according to claim 1 or 2, characterized in that, The 1×Ca-free 2+ / Mg 2+ PBS was prepared from ultrapure water with a resistivity ≥18 MΩ·cm.

4. The synaptic heterogeneity analysis device based on multicolor flow cytometry according to any one of claims 1-3, characterized in that, The sheath fluid is used after being filtered through a 0.22-0.25 µm filter membrane; Preferably, the carrier buffer solution is used after being filtered through a 0.22-0.25 µm filter membrane.

5. The synaptic heterogeneity analysis device based on multicolor flow cytometry according to any one of claims 1-4, characterized in that, The fluorescent markers include: neurotransmitter type markers and functional status markers; The neurotransmitter type marker is the synaptophysin-tdTomato fusion protein, with an excitation wavelength of 561 nm and an emission wavelength of 610 / 20 nm; the functional state marker is FM4-64, with an excitation wavelength of 488 nm and an emission wavelength of 620 / 30 nm.

6. The synaptic heterogeneity analysis device based on multicolor flow cytometry according to any one of claims 1-5, characterized in that, The logic gate-double exponential decay compensation algorithm settings include: (a) Single-stain microsphere calibration: Thermo UltraRainbow 0.5 µm microspheres were used to single stain two types of fluorescence, 10,000 events were collected, and a 2×2 spectral overlap matrix was established; (b) Double exponential decay compensation algorithm: Let I raw I represents the original fluorescence intensity. corr To compensate for the strength, then, ； Parameters k1-k4 were obtained by Levenberg-Marquardt nonlinear fitting, and the residual crosstalk after compensation was <5%. (c) Logic gate sorting: P1: FSC-SSC elliptical gate, delineating synaptosome groups; P2: vGlut1 / GAD2 / DAT×AI34, neurotransmitter gating, threshold set to mean + 3 SD for single-staining positive microspheres; P3 / P4: FM4-64, a functional gate with threshold values ​​at the 20% of strongest and weakest fluorescence intensities; Sorting P3 / P4: P3: vGlut1 + / FM4-64 + Activates glutamatergic energy; P4: vGlut1 + / FM4-64 - Resting glutamate can.

7. A method for analyzing synaptic heterogeneity based on multicolor flow cytometry, characterized in that, The analytical method includes: (1) Collect synaptosome samples from brain tissue; (2) Using the BD FACS fusion dual-laser platform, the FSC-SSC coordinates were first located with standard 0.5 µm polystyrene microspheres, and then the collected synaptic body samples were loaded. (3) FSC-SSC first sets up a gate to exclude noise and debris; (4) Dual-channel acquisition at 488 nm / 561 nm with real-time compensation; (5) The logic gate is used for two-way sorting. The two collection tubes are connected to P3 / P4 respectively to obtain the sorted synapses.

8. The synaptic heterogeneity analysis method based on multicolor flow cytometry according to claim 7, characterized in that, In step (4), the parameters for real-time compensation are set as follows: double exponential decay compensation, residual crosstalk <5%.

9. The synaptic heterogeneity analysis method based on multicolor flow cytometry according to claim 7 or 8, characterized in that, In step (5), the parameters of the logic gate two-way sorting are set as follows: P1 elliptical gate FSC-SSC delineates the synaptosome; P2 neurotransmitter gate threshold is set to mean + 3 SD of single-stained positive microspheres; P3 / P4 functional gate thresholds are respectively set to 20% of the strongest and 20% of the weakest fluorescence intensity of FM4-64.

10. The application of the synaptic heterogeneity analysis device based on multicolor flow cytometry according to any one of claims 1-6 or the synaptic heterogeneity analysis method based on multicolor flow cytometry according to any one of claims 7-9 in synaptic detection.

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