Lysosome extraction method

Abstract

The invention provides a lysosome extraction method. The lysosome extraction method comprises the following steps: (1) extracting cells; (2) tissue extraction; (3) crude separation of lysosome; and (4) separating the lysosome by magnetic beads. Through EDTA-free digestion optimization, lysis solution and buffer solution formula optimization, a filter membrane interception-magnetic bead purification combined technology, a double-antibody synergistic capture strategy and a competitive mild elution system, the four core problems of low purity, large activity damage, tedious operation and strong equipment dependence in a traditional lysosome separation technology are systematically solved. Experimental data show that the lysosome yield and functional integrity of the method are obviously superior to those of a density gradient centrifugation method, and the method is suitable for trace samples and conventional equipment and has high conversion value. In the future, the technical barrier can be further optimized through automation equipment integration and multi-dimensional function verification, and the application of the method in basic research and clinical diagnosis is promoted.

Description

Technical Field

[0001] The invention relates to a lysosome extraction method. Background Art

[0002] With the continuous development of multidisciplinary research methods and omics technologies such as molecular cell biology, organelle imaging, and dynamic tracing observations, the study of organelles has become increasingly in-depth. Obtaining intact, highly pure lysosomes is an important foundation for studying lysosomal microstructure, homeostatic regulation, and the localization of related biomolecules. The improvement and application of lysosomal separation and purification technologies are one of the innovative trends in biotechnology. Commonly used lysosomal separation and purification techniques include differential centrifugation, density gradient centrifugation, and fluorescence-assisted organelle sorting.

[0003] Current differential centrifugation methods separate organelles based on differences in particle sedimentation velocity. However, the difference in sedimentation coefficient between lysosomes and other organelles (such as mitochondria and peroxisomes) is small, resulting in low lysosomal purity after separation. Furthermore, the separation process requires multiple centrifugation and washing steps, which can cause lysosomes to rupture or be lost during repeated manipulations. For example, multiple low-speed centrifugations are required to remove cell debris, and excessive centrifugation can disrupt lysosomal membrane structure. Flow cytometry using fluorescence sorting equipment (such as flow cytometers) requires high-pressure sheath fluid to propel cells, which can cause lysosomal membrane rupture. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a lysosome extraction method.

[0005] The present invention provides a lysosome extraction method, comprising the following steps: (1) Cell extraction: Digest the cells with EDTA-free trypsin. After terminating the digestion, mix the cell pellet with the lysis buffer and repeatedly pipette and lyse on ice for 30 min in an ice bath. Use trypan blue staining to monitor the cell lysis rate in real time, dynamically adjust the lysis time, and regulate the lysis rate to 85% ± 5% in real time to obtain a mixture of cell extracts. (2) Tissue extraction: Mix the tissue and lysate, add yttrium-stabilized zirconia beads, and grind at 4°C to obtain a tissue extract suspension; (3) Lysosome crude separation: The cell extract mixture of step (1) or the tissue extract suspension of step (2) is filtered through a Whatman 934 AH glass fiber filter paper membrane with a diameter of 3 μm to 1.5 μm in series, and the filtered liquid is retained and centrifuged at 18,000 to 22,000 g for 15 to 25 minutes. The precipitate is collected and resuspended in a basal buffer for lysosome extraction to obtain a lysosome resuspension; (4) Separation of lysosomes using magnetic beads: The lysosome resuspension obtained in step (3) was mixed with the antibody-coupled magnetic beads, incubated on a shaker for 30 minutes, and placed in a KingFisher Flex magnetic bead purification system for binding and washing. The specifically bound lysosomes were then eluted from the magnetic beads with an elution buffer and immediately neutralized with a neutral preservation solution after elution.

[0006] Preferably, in step (1), the cells include adherent cells, suspension cells, primary cells and stem cells.

[0007] Preferably, in step (1) and step (2), the formula of the lysate is: 0.25M sucrose + 0.15M sorbitol + 10mM HEPES buffer + 10mM KCl + 1mM MgCl2 + 1mM phenylmethylsulfonyl fluoride + 1-10μM protease inhibitor A + 0.1g / L digitonin.

[0008] Preferably, in step (2), when the tissue is liver tissue, 1 mm yttrium-stabilized zirconia beads are added, and the grinding method is: 6×30 s pulse grinding, with 1 min cooling interval; When the tissue is brain tissue, 0.5 mm yttrium-stabilized zirconia beads are added, and the grinding method is: 4×20 s pulses with 1 min cooling intervals.

[0009] Preferably, in step (3), the formula of the lysosome extraction-specific basic buffer is: 20mM HEPES buffer + 20mM MES buffer + 120mM NaCl + 250mM sucrose + 1g / L polyvinylpyrrolidone-40 + 0.1mM CaCl2 + 0.5-1mM MgCl2.

[0010] Preferably, in step (4), the antibody consists of LAMP1 antibody and LAMP2 antibody, and the volume ratio of the two is 1:3-3:1; preferably, the volume ratio of the two is 1:1.

[0011] Preferably, in step (4), the preparation method of the antibody-coupled magnetic beads is: coupling the activated magnetic beads with the antibody, and then performing a blocking treatment; Preferably, the activated magnetic beads and the antibody mixture are incubated with rotation at 37° C. for 1.5-4 hours, and then washed with PBS containing 0.05% by volume Tween-20 to remove unbound antibodies.

[0012] Preferably, in step (4), after being placed in the KingFisher Flex magnetic bead purification system, the processing procedure is: binding for 5 minutes → washing 3 times.

[0013] Preferably, in step (4), the eluent is a glycine-HCl buffer solution at pH 2.5.

[0014] Preferably, in step (4), the neutral preservation solution is a HEPES buffer solution with a pH of 7.4; the formula of the HEPES buffer solution is as follows: 35-50 mM HEPES + 150-250 mM sucrose.

[0015] The beneficial effects of the present invention are as follows: 1. In the cell extraction step of the prior art, the trypsin digestion time is too long (3 minutes), resulting in lysosomal membrane rupture, and EDTA chelates Ca 2+ and Mg 2+ It will weaken the adsorption efficiency of negatively charged organelles.

[0016] In the cell extraction step of the present invention, EDTA-free pancreatic enzyme is used, and the digestion time gradient (0.5 / 1 / 2 / 3 minutes) is optimized by acid phosphatase (ACP) activity detection, and the digestion time is shortened to 1 minute to avoid EDTA chelation of Ca 2+ and Mg 2+ This results in a decrease in the adsorption efficiency of negatively charged organelles. Using the method of the present invention, the lysosomal marker enzyme (acid phosphatase, ACP) increased by 35.7% at 1 minute compared to 3 minutes, significantly reducing the risk of membrane rupture.

[0017] 2. In the cell extraction step of the prior art, traditional lysis methods such as ultrasound or high-pressure disruption are used, which can easily damage the lysosomal membrane and lead to the loss of enzyme activity.

[0018] In the cell extraction step of the present invention, the lysis solution formula is optimized, sucrose and sorbitol are used to coordinately regulate the osmotic pressure, and Mg is introduced in advance. 2+ Locks lysosomal membrane stability; adds digitonin to target plasma membrane lysis; uses a HEPES buffer system to ensure downstream immunomagnetic bead binding efficiency; and protease inhibitor A specifically inhibits lysosomal acid proteases.

[0019] Furthermore, on-ice lysis is used instead of high-energy disruption. Trypan blue staining is combined with real-time monitoring of lysis rates, allowing for dynamic adjustment of lysis time to balance lysis efficiency and membrane integrity. Using this method, the lysosomal membrane damage rate is reduced to below 15% (compared to 30% with existing methods).

[0020] 3. In the tissue extraction step of the existing technology, traditional grinding beads are used, which will release metal ions and interfere with subsequent experiments.

[0021] The present invention uses a tissue-specific grinding strategy (zirconia bead size-pulse parameter linkage optimization, such as 1mm for liver tissue and 0.5mm for brain tissue, 6×30s pulses with intermittent cooling for liver tissue; 4×20s pulses with intermittent cooling for brain tissue) to achieve a liver tissue lysis efficiency of over 85% and a brain tissue lysis efficiency of over 75%, an improvement of 35-40% over traditional methods, and a lysosome recovery rate of over 90% (verified by density gradient centrifugation combined with LAMP1 immunomagnetic bead sorting), significantly superior to existing technologies.

[0022] 4. In the prior art, in the crude lysosome separation step, a single-stage filter membrane (eg, 1.5 μm) is used, which is insufficient for retaining mitochondria (0.5-1.0 μm), and the density gradient centrifugation takes a long time (several hours).

[0023] In the crude lysosome separation step of the present invention, a series of 3μm+1.5μm filter membranes are used to grade and retain cell nuclei (3-30μm), cell fragments and mitochondria (retention rate>95%); at the same time, combined with charge gradient adsorption (addition of Ca 2+ / Mg 2+ The lysosome extraction special basic buffer is used to enhance the filter membrane adsorption efficiency of negatively charged organelles (mitochondria, endoplasmic reticulum), making the lysosome (0.025~0.8μm) pass rate >95%.

[0024] The lysosome extraction step of this invention features an optimized buffer formulation. This triple design, combining HEPES-MES dual buffering, sucrose isotonicity, and a membrane-stabilizing polymer, completely resolves the precipitation and enzyme activation issues associated with traditional KPBS. Furthermore, CaCl2 and MgCl2 enhance the adsorption of negatively charged organelles (mitochondria / endoplasmic reticulum), improving membrane separation efficiency. Polyvinylpyrrolidone-40 encapsulates the lysosomes, reducing mechanical damage and maintaining lysosomal membrane integrity.

[0025] 5. In the existing technology, single antibody magnetic bead sorting (such as only LAMP1) is used, with a low capture rate (70%), and traditional elution buffers (such as low pH) easily damage the lysosomal membrane (rupture rate > 50%).

[0026] In the present invention, dual antibodies (LAMP1:LAMP2 volume ratio = 1:1) were used for synergistic capture to increase the lysosome recovery rate to 95%. The KingFisher Flex program was used: binding for 5 minutes → washing 3 times, and glycine-HCl buffer (pH 2.5) was used to gently elute the antigen-antibody binding, optimizing the sorting efficiency and maintaining lysosomal membrane integrity at >85%.

[0027] 6. In the prior art, the total operation time is 4-6 hours.

[0028] In the present invention, the KingFisher Flex magnetic bead purification system is used, which shortens the total operation time to 2 hours (4-6 hours for traditional methods), eliminates the need for an ultracentrifuge, and increases the total protein yield by 2-3 times (e.g., from 150 μg to 320 μg for primary cells).

[0029] 7. In the existing technology, density gradient centrifugation is used to process micro samples (such as primary cells), which has low processing efficiency (the total protein yield is only 150μg) and is highly equipment-dependent.

[0030] The method of the present invention is suitable for adherent cells, suspension cells, primary cells and stem cells, and the total protein yield is increased by 2 to 3 times compared with the existing technology (for example, primary cells from 150 μg to 320 μg); and the method of the present invention relies on conventional equipment (such as centrifuges, magnetic bead purification systems), without the need for ultracentrifugation or fluorescence sorters, thus lowering the threshold for use.

[0031] The present invention systematically solves the four core problems of traditional lysosome separation technology, namely low purity, large activity damage, cumbersome operation, and strong equipment dependence, through EDTA-free digestion optimization, lysis solution and buffer solution formulation optimization, filter membrane retention-magnetic bead purification combination technology, double antibody collaborative capture strategy and competitive mild elution system. Experimental data show that its lysosome yield and functional integrity are significantly better than those of density gradient centrifugation, and it is suitable for trace samples and conventional equipment, with high conversion value. In the future, the integration of automated equipment and multi-dimensional functional verification can be used to further optimize technical barriers and promote its application in basic research and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 To test the purity of lysosomes. DETAILED DESCRIPTION

[0033] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0034] Reagents used in the present invention: (1) EDTA-free trypsin digestion solution: Gibco product number 15050-065.

[0035] (2) DMEM complete medium, Gibco product number 12491015.

[0036] (3) Yttrium-stabilized zirconia beads (core component is high-purity zirconia (ZrO2), Pingxiang Jinrui New Materials Co., Ltd., item number 1314-23-4.

[0037] (4) Dynabeads M-270, Invitrogen, Cat. No. 14305D.

[0038] (5) LAMP1 antibody, Invitrogen catalog number 14-1071-82.

[0039] (6) LAMP2 antibody, Abcam Catalog No. ab25631.

[0040] The lysosome extraction method of the present invention comprises the following steps: 1. Cell Extraction: Cells were digested with 1 ml of EDTA-free trypsin digestion buffer to avoid weakening the adsorption of negatively charged organelles. Overdigestion can easily lead to lysosomal membrane rupture, especially since lysosomal membranes contain a large number of enzyme-sensitive glycoproteins. A time gradient experiment (0.5 / 1 / 2 / 3 minutes) combined with lysosomal marker enzyme (ACP) activity assay revealed that the lysosomal marker enzyme (acid phosphatase, ACP) concentration at 1 minute was 95 ± 2.8 U / mg, a 35.7% increase from 70 ± 3.5 U / mg at 3 minutes, indicating an optimal digestion time of 1 minute. Digestion was terminated by adding 3 ml of complete DMEM medium and washed three times with PBS (1000 g for 5 minutes). Cell sample amounts are shown in Tables 2-5. Cell pellets were then added to 400 μl of lysis buffer and lysed on ice for 30 minutes by repeated pipetting and on ice. Cell lysis was monitored using trypan blue staining in real time, and the lysis time was dynamically adjusted to maintain a lysis rate of 85% ± 5%. A mixed cell extract was obtained.

[0041] The lysis buffer formula is: 0.25M sucrose + 0.15M sorbitol + 10mM HEPES buffer + 10mM KCl + 1mM MgCl2 + 1mM phenylmethylsulfonyl fluoride + 1-10μM protease inhibitor A + 0.1g / L digitonin.

[0042] In the lysis solution formula, sucrose + sorbitol are used to maintain the osmotic pressure balance of the organelles, and Mg is introduced in advance. 2+ Locks lysosomal membrane stability; adds digitonin to target plasma membrane lysis; uses a HEPES buffer system to ensure downstream immunomagnetic bead binding efficiency; and protease inhibitor A specifically inhibits lysosomal acid proteases.

[0043] 2. Tissue Extraction: Wash 30-90 mg of fresh tissue with PBS. After washing, add lysis buffer (300-600 μl per 30 mg) (the lysis buffer recipe is the same as in step 1). Add appropriate amounts of yttrium-stabilized zirconia beads of varying sizes (e.g., 1 mm for liver tissue and 0.5 mm for brain tissue) to improve lysis efficiency for different tissue types. Grind in a grinder at 4°C (6 x 30 s pulses with 1-min cooling intervals for liver tissue; 4 x 20 s pulses with 1-min cooling intervals for brain tissue) until the tissue lysis efficiency reaches >85% and >75% for brain tissue. Prepare a tissue extract suspension.

[0044] 3. Lysosome crude separation: Depending on the sample type, filter the cell extract mixture from step 1 or the tissue extract mixture from step 2 through a 3μm-1.5μm Whatman 934 AH glass fiber filter paper membrane in series. The glass fiber structure of the Whatman 934 AH filter membrane can adsorb negatively charged cell debris (such as mitochondrial membrane containing cardiolipin), while lysosomes pass through the filter membrane due to the positive charge of their surface glycoproteins. The filter retains target organelles (lysosomes, 0.025μm-0.8μm), removing nuclei (3-30μm), cell debris, heavy mitochondria (1.5-3.0μm), and endoplasmic reticulum (40-50μm). The filter paper is discarded, and the filtered liquid is retained, leaving behind mitochondria (0.5-1.0μm), ribosomes (15-25nm), centrosomes (0.3-0.5μm), catalsomes (0.2-1.5μm), and lysosomes (0.025μm-0.8μm). Nanoparticle tracking analysis showed that the 3μm-1.5μm tandem filter membrane retained >95% of particles larger than 1.5μm, with a catalsome retention rate of less than 5% (compared to 15% with traditional gradient centrifugation). Further centrifugation at 18,000–22,000 g for 15–25 minutes (4°C) will pellet the lysosomes (density 1.10–1.12 g / cm³). The pellet is then resuspended in lysosome extraction basal buffer, increasing the adsorption of negatively charged organelles and introducing a charge gradient for subsequent filtration and separation. Compared to traditional PBS, lysosome extraction basal buffer maintains lysosomal membrane stability by regulating ionic strength.

[0045] Lysosome extraction-specific basic buffer: 20mM HEPES buffer + 20mM MES buffer + 120mM NaCl + 250mM sucrose + 1g / L polyvinylpyrrolidone-40 + 0.1mM CaCl2 + 0.5-1mM MgCl2.

[0046] This lysosome extraction-specific basal buffer utilizes a four-pronged design: HEPES-MES dual buffering, sucrose isotonicity, polyvinylpyrrolidone-40 for membrane stability, and precise ion control polymers. This design completely eliminates the precipitation and enzyme activation issues associated with traditional KPBS. The basal buffer maintains lysosomal membrane stability; polyvinylpyrrolidone-40 coats the lysosomes, minimizing mechanical damage and preserving membrane integrity; and CaCl2 and MgCl2 enhance the adsorption of negatively charged organelles (mitochondria / endoplasmic reticulum), improving membrane separation efficiency.

[0047] 4. Preparation of Antibody-coupled Magnetic Beads: (1) Activation of magnetic beads: Take 1 mg of carboxyl magnetic beads (such as Dynabeads M-270) and wash them three times with 1 ml of 100 mM 2-(N-Morpholino)ethanesulfonic Acid Buffer (MES) buffer; add 1 ml of MES solution containing 10 mM 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 5 mM N-hydroxysuccinimide (NHS) and activate them by shaking at room temperature for 25-40 minutes; discard the supernatant, add 1 ml of MES to wash the magnetic beads, and then add 500 μl of MES to resuspend the magnetic beads.

[0048] (2) Antibody coupling: Take 100 μl of LAMP1 antibody (Invitrogen, 14-1071-82) and 100 μl of LAMP2 antibody (Abcam, ab25631), mix and use directly. Mix the magnetic beads from step (1) with 200 μl of the antibody mixture to a concentration of 5 mg / ml. Incubate with rotation at 37°C for 1.5-4 hours, magnetically separate, and wash three times with PBS containing 0.05% Tween-20 by volume to remove unbound antibody.

[0049] Experiments were conducted using LAMP1:LAMP2 antibody mixtures at volume ratios of 1:1, 1:2, 2:1, 1:3, and 3:1, and the amount of unbound LAMP protein in the supernatant was determined by ELISA. The results showed that the highest lysosomal recovery rate (95%) was achieved when the LAMP1:LAMP2 antibody mixture was 1:1. Compared to single antibody methods (traditional methods using only LAMP1), dual antibody methods can increase lysosomal capture efficiency from 70% to 95%.

[0050] (3) Blocking treatment: Resuspend the coupled magnetic beads in 500 μl of PBS containing 5% BSA by volume and rotate for 1-2 hours at room temperature. Discard the supernatant and wash thoroughly three times with 500 μl of PBS buffer. Finally, resuspend the magnetic beads in 500 μl of PBS buffer and store at 4°C.

[0051] 5. Magnetic Bead Isolation of Lysosomes: Add the lysosome resuspension obtained in Step 3 to the antibody-coupled magnetic beads obtained in Step 4. Incubate on a shaker for 30 minutes to allow the beads to bind to the antigen through the antibody. Place the beads in a KingFisher Flex Magnetic Purification System (Procedure: Bind 5 minutes → Wash 3 times). Collect the lysosome suspension by centrifugation (1000g x 2 minutes). Remove the magnetic bead complex and gently elute it with 1 ml of 100 mM glycine-HCl (pH 2.5) elution buffer. Shake on ice for 30-60 seconds (brief acidic exposure to avoid membrane damage) to elute specifically bound lysosomes from the beads. Immediately neutralize with 200 μl of neutral preservation buffer (35-50 mM HEPES + 150-250 mM sucrose, pH 7.4) and gently shake to mix (pH 2.5 to 7.4, within 10 seconds). Lysosomes themselves tolerate an acidic environment (intralysosomal pH approximately 4.5-5.0), and brief exposure to pH 2.5 (combined with rapid neutralization) poses a low risk of damaging the membrane structure.

[0052] 6. Detection of lysosomal integrity: The functional integrity of lysosomal enzymes was reflected by detecting the activity of the lysosomal marker enzyme acid phosphatase. The extracted lysosomes were incubated with the substrate p-nitrophenyl phosphate (pNPP) at 37°C for 30 minutes, and the absorbance at 405 nm was measured. The results showed that compared with the conventional density gradient centrifugation method (Method A), the ACP activity of lysosomes extracted by the method of the present invention (Method B) was (25.3±1.2 U / mg), which was significantly higher than that of Method A (18.7±0.9 U / mg) ( P <0.01).

[0053] Table 1 shows the comparison of technical effects (compared with traditional density gradient centrifugation).

[0054] Table 1 index Traditional methods The present invention Improvement effect ACP activity retention 18.7±0.9U / mg 25.3±1.2U / mg Activity increased by 35% Total protein yield 150~500μg 320~850μg Yield increased by 2~3 times Operation time 4~6 hours ≤2 hours Efficiency increased by more than 50% Device Dependency ultracentrifuge Conventional centrifuge + magnetic bead system Cost reduction of 60% Example 1 The lysosome extraction method of the present embodiment comprises the following steps: 1. Cell Extraction: Digest the cells with 1 ml of EDTA-free trypsin for 1 minute. Stop the digestion with 3 ml of complete DMEM medium and wash three times with PBS (1000 g for 5 minutes). See Tables 2-5 for cell sample amounts. Add 400 μl of lysis buffer to the cell pellet and repeatedly pipette and lyse on ice for 30 minutes in an ice bath. Monitor the cell lysis rate in real time using trypan blue staining, dynamically adjust the lysis time, and maintain the lysis rate at 85% to obtain a mixed cell extract.

[0055] The lysis buffer formula is: 0.25M sucrose + 0.15M sorbitol + 10mM HEPES buffer + 10mM KCl + 1mM MgCl2 + 1mM phenylmethylsulfonyl fluoride + 5μM protease inhibitor A + 0.1g / L digitonin.

[0056] 2. Lysosome crude isolation: Depending on the sample type, filter the extract mixture obtained in step 1 through a Whatman 934 AH glass fiber filter paper membrane with a diameter of 3 μm to 1.5 μm in series. Discard the filter paper and retain the filtered liquid. Centrifuge it at 20,000 g for 20 minutes (4°C). Collect the precipitate and resuspend it in 1 ml of lysosome extraction buffer.

[0057] Lysosome extraction-specific basic buffer: 20mM HEPES buffer + 20mM MES buffer + 120mM NaCl + 250mM sucrose + 1g / L polyvinylpyrrolidone-40 + 0.1mM CaCl2 + 0.5mM MgCl2.

[0058] 3. Preparation of Antibody-Conjugated Magnetic Beads: (1) Activation of magnetic beads: Take 1 mg of carboxyl magnetic beads Dynabeads M-270, wash them three times with 1 ml of 100 mM MES buffer, add 1 ml of MES containing 10 mM EDC and 5 mM NHS, activate them at room temperature with shaking for 30 min, discard the supernatant, add 1 ml of MES to wash the magnetic beads, and then add 500 μl of MES to re-dissolve the magnetic beads.

[0059] (2) Antibody coupling: Take 100 μl of LAMP1 antibody and 100 μl of LAMP2 antibody, mix and use directly. Mix the magnetic beads from step (1) with 200 μl of the antibody mixture to a concentration of 5 mg / ml. Incubate with rotation at 37°C for 2 hours, magnetically separate, and wash three times with PBS containing 0.05% Tween-20 by volume to remove unbound antibody.

[0060] (3) Blocking treatment: Resuspend the coupled magnetic beads in 500 μl of PBS containing 5% BSA by volume and rotate for 1 hour at room temperature. Discard the supernatant and wash thoroughly three times with 500 μl of PBS buffer. Finally, resuspend the magnetic beads in 500 μl of PBS buffer and store at 4°C.

[0061] 4. Magnetic Bead Isolation of Lysosomes: Add the lysosome resuspension obtained in Step 2 to the antibody-coupled magnetic beads obtained in Step 3. Incubate on a shaker for 30 minutes. Place in a KingFisher Flex Magnetic Purification System (Procedure: Bind 5 minutes → Wash 3 times). Collect the lysosome suspension by centrifugation (1000g x 2 minutes). Remove the magnetic bead complex and add 1 ml of 100 mM glycine-HCl (pH 2.5) elution buffer. Elute on ice for 60 seconds with shaking. Immediately after elution, transfer to a pre-chilled neutralization tube (containing 200 μl of neutral preservation buffer HEPES buffer, pH 7.4, 35 mM HEPES + 150 mM NaCl) for neutralization. Gently shake to mix (pH 2.5 → 7.4, within 10 seconds).

[0062] 5. Lysosomal integrity: Detecting the activity of the lysosomal marker enzyme acid phosphatase reflects the functional integrity of lysosomal enzymes. Incubate the extracted lysosomes with the substrate p-nitrophenyl phosphate at 37°C for 30 minutes, and measure the absorbance at 405 nm.

[0063] 6. The expression of lysosomal marker LAMP2 was detected by Western blot to test the purity of the extraction of the present invention. The results showed that compared with the conventional density gradient centrifugation method (method A), the lysosomes extracted by the method of the present invention (method B) were of higher purity and had higher practical value (see Figure 1 ).

[0064] Figure 1 To test the purity of lysosomes.

[0065] By optimizing the lysis, isolation, and purification steps, this method is widely applicable to adherent, suspension, and primary cells. The following data are experimentally analyzed for extraction efficacy in different cell types (total protein yield was determined by the BCA assay, and lysosomal functional integrity was assessed by ACP activity).

[0066] (1) Adherent cells (taking HeLa cells as an example): Cell type: HeLa (human cervical cancer cells); Sample size: 1×10^7 cells / group.

[0067] The experimental results are shown in Table 2.

[0068] Table 2 index Density gradient centrifugation Method of the present invention Total protein yield (μg) 500±30 850±50 ACP activity (U / mg) 18.7±0.9 24.8±1.5 (2) Suspension cells (taking Jurkat cells as an example): Cell type: Jurkat (human T lymphocytes); Sample size: 5×10^6 cells / group.

[0069] The experimental results are shown in Table 3.

[0070] Table 3 index Density gradient centrifugation Method of the present invention Total protein yield (μg) 250±20 420±30 ACP activity (U / mg) 15.2±0.8 20.5±1.0 (3) Primary cells (taking mouse liver cells as an example): Cell type: primary mouse hepatocytes; Sample size: 2×10^6 cells / group.

[0071] The experimental results are shown in Table 4.

[0072] Table 4 index Density gradient centrifugation Method of the present invention Total protein yield (μg) 150±15 320±25 ACP activity (U / mg) 12.0±0.7 18.2±0.9 (4) Special cell types (taking stem cells as an example): Cell type: stem cells; Sample size: 1×10^7 cells / group.

[0073] The experimental results are shown in Table 5.

[0074] Table 5 index Density gradient centrifugation Method of the present invention Total protein yield (μg) 350±25 600±40 ACP activity (U / mg) 14.5±0.9 19.8±1.0 Comparative Example 1 Lysis Solution Formula Optimization Experiment The only difference between this comparative example and Example 1 is that the formula of the lysate is different. The remaining steps and parameters are the same as those in Example 1.

[0075] Control group: Lysis buffer A (formula in Example 1, denoted as "Standard A"): 0.25 M sucrose + 0.15 M sorbitol + 10 mM HEPES buffer + 10 mM KCl + 1 mM MgCl2 + 1 mM phenylmethylsulfonyl fluoride + 5 μM protease inhibitor A + 0.1 g / L digitonin.

[0076] The experimental group design is shown in Table 6.

[0077] Table 6 Comparative Example Lysis buffer adjustment Design basis 1-1 Remove digitonin Verify the necessity of membrane permeabilization agent 1-2 The concentration of digitonin increased to 0.3 g / L Investigating the damage of high concentration detergents to membrane structure 1-3 Removal of phenylmethylsulfonyl fluoride and protease inhibitor A Verify the protection of enzyme activity by protease inhibitors 1-4 Halve the sucrose / sorbitol concentration Testing osmotic protection 1-5 Replace digitonin with 0.01% TritonX-100 by volume Comparing the mildness of different detergents The ACP leakage rate (%) and LAMP2 recovery rate (%) of different groups were measured as follows: ACP leakage rate: The lysosomal suspension was centrifuged and the supernatant (free ACP) and precipitate (membrane-bound ACP) were measured separately: leakage rate = (supernatant ACP activity / total ACP activity) × 100%.

[0078] LAMP2 recovery rate: Western blot quantification, with the standard group as 100%: recovery rate = (gray level of LAMP2 band in control group / gray level of standard group) × 100%.

[0079] The experimental results are shown in Table 7.

[0080] Table 7 Group ACP leakage rate (%) LAMP2 recovery rate (%) Standard A 15.2 ± 1.8 100.0 ± 5.3 1-1 (-Digitonin) 42.6 ± 3.5 35.7 ± 4.1 1-2 (0.3g / L digitonin) 68.3 ± 4.2 18.9 ± 2.7 1-3 (-Protease Inhibitor A) 37.4 ± 2.9 52.1 ± 3.8 1-4 (low osmotic pressure) 54.2 ± 3.1 41.5 ± 3.2 1-5 (Triton X-100) 89.5 ± 5.1 9.3 ± 1.5 Therefore, the digitonin concentration must be strictly controlled at 0.1g / L: too low a concentration results in incomplete lysis, while too high a concentration can damage the membrane structure. The absence of osmotic stabilizers (sucrose / sorbitol) can lead to lysosomal edema and rupture. Protease inhibitors are crucial for maintaining enzyme activity. Digitonin concentration, osmotic balance, and protease inhibitors are irreplaceable.

[0081] Comparative Example 2 Lysosome Extraction Special Basic Buffer Formula Optimization Experiment The only difference between this comparative example and Example 1 is that the formula of the basal buffer solution for lysosome extraction is different. The remaining steps and parameters are the same as those in Example 1.

[0082] Control group: Lysosome extraction-specific basic buffer B (the recipe in Example 1, recorded as "Standard B"): 20 mM HEPES buffer + 20 mM MES buffer + 120 mM NaCl + 250 mM sucrose + 1 g / L polyvinylpyrrolidone-40 + 0.1 mM CaCl2 + 0.5 mM MgCl2.

[0083] The experimental group design is shown in Table 8.

[0084] Table 8 Comparative Example Buffer adjustment Design basis 2-1 Remove polyvinylpyrrolidone-40 Verify the effect of anti-adsorbent 2-2 Add 1 mM EDTA Investigating the effect of chelating agents on membrane stability 2-3 Remove Ca²⁺ / Mg²⁺ Verifying the protection of membrane proteins by divalent ions 2-4 Sucrose concentration was reduced to 50 mM Testing for hypotonic environmental damage The ACP leakage rate (%) and LAMP2 recovery rate (%) were determined using the same method as above.

[0085] The experimental results are shown in Table 9.

[0086] Table 9 Group ACP leakage rate (%) LAMP2 recovery rate (%) Magnetic bead aggregation phenomenon Standard B 16.8 ± 2.1 100.0 ± 6.2 none 2-1 (-Polyvinylpyrrolidone-40) 38.5 ± 3.2 47.3 ± 4.5 Severe aggregation of magnetic beads 2-2 (+EDTA) 61.4 ± 4.8 22.7 ± 3.1 Partial aggregation 2-3 (-Ca²⁺ / Mg²⁺) 45.2 ± 3.7 58.9 ± 4.2 Slight aggregation 2-4 (low sucrose) 73.6 ± 5.3 15.4 ± 2.0 No gathering Therefore, polyvinylpyrrolidone-40 prevents nonspecific adsorption of magnetic beads, and its absence results in a more than 50% decrease in yield. EDTA chelates Ca²⁺ / Mg²⁺, triggering membrane protein shedding and significantly increasing ACP leakage. Maintaining osmotic pressure (250mM sucrose) is essential for membrane integrity. The anti-adsorption properties of polyvinylpyrrolidone-40 and the membrane stabilization by divalent ions are among the innovative features of this invention.

[0087] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A lysosome extraction method, characterized in that: The following steps are involved: (1) Cell extraction: Digest the cells with EDTA-free trypsin. After terminating the digestion, mix the cell pellet with the lysis buffer and repeatedly pipette and lyse on ice for 30 min in an ice bath. Use trypan blue staining to monitor the cell lysis rate in real time, dynamically adjust the lysis time, and regulate the lysis rate to 85% ± 5% in real time to obtain a mixture of cell extracts. (2) Tissue extraction: Mix the tissue and lysate, add yttrium-stabilized zirconia beads, and grind at 4°C to obtain a tissue extract suspension; (3) Lysosome crude separation: The cell extract mixture of step (1) or the tissue extract suspension of step (2) is filtered through a Whatman 934 AH glass fiber filter paper membrane with a diameter of 3 μm to 1.5 μm in series, and the filtered liquid is retained and centrifuged at 18,000 to 22,000 g for 15 to 25 minutes. The precipitate is collected and resuspended in a basal buffer for lysosome extraction to obtain a lysosome resuspension; (4) Separation of lysosomes using magnetic beads: The lysosome resuspension obtained in step (3) was mixed with the antibody-coupled magnetic beads, incubated on a shaker for 30 minutes, and placed in a KingFisher Flex magnetic bead purification system for binding and washing. The specifically bound lysosomes were then eluted from the magnetic beads with an elution buffer and immediately neutralized with a neutral preservation solution after elution.

2. A lysosome extraction method according to claim 1, characterized in that: In step (1), the cells include adherent cells, suspension cells, primary cells and stem cells.

3. A lysosome extraction method according to claim 1, characterized in that: In step (1) and step (2), the formula of the lysis solution is: 0.25M sucrose + 0.15M sorbitol + 10mM HEPES buffer + 10mM KCl + 1mM MgCl2 + 1mM phenylmethylsulfonyl fluoride + 1-10μM protease inhibitor A + 0.1g / L digitonin.

4. A lysosome extraction method according to claim 1, characterized in that: In step (2), when the tissue is liver tissue, 1 mm yttrium-stabilized zirconia beads are added, and the grinding method is: 6 × 30 s pulse grinding, with 1 min cooling interval; When the tissue is brain tissue, 0.5 mm yttrium-stabilized zirconia beads are added, and the grinding method is: 4×20 s pulses with 1 min cooling intervals.

5. A lysosome extraction method according to claim 1, characterized in that: In step (3), the formula of the lysosome extraction special basic buffer is: 20mM HEPES buffer + 20mM MES buffer + 120mM NaCl + 250mM sucrose + 1g / L polyvinylpyrrolidone-40 + 0.1mM CaCl2 + 0.5-1mM MgCl2.

6. A lysosome extraction method according to claim 1, characterized in that: In step (4), the antibody consists of LAMP1 antibody and LAMP2 antibody, and the volume ratio of the two is 1:3-3:1; preferably, the volume ratio of the two is 1:

1.

7. A lysosome extraction method according to claim 1 or 6, characterized in that: In step (4), the method for preparing the antibody-coupled magnetic beads is as follows: coupling the activated magnetic beads with the antibody, and then performing a blocking treatment; Preferably, the activated magnetic beads and the antibody mixture are incubated with rotation at 37° C. for 1.5-4 hours, and then washed with PBS containing 0.05% by volume Tween-20 to remove unbound antibodies.

8. A lysosome extraction method according to claim 1, characterized in that: In step (4), after placing in the KingFisher Flex magnetic bead purification system, the processing procedure is: binding for 5 minutes → washing 3 times.

9. A lysosome extraction method according to claim 1, characterized in that: In step (4), the eluent is a glycine-HCl buffer solution at pH 2.

5.

10. A lysosome extraction method according to claim 1, characterized in that: In step (4), the neutral preservation solution is a HEPES buffer solution with a pH of 7.4; the formula of the HEPES buffer solution is as follows: 35-50 mM HEPES + 150-250 mM sucrose.

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