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Jan 2022

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A Robust Nanoparticle-based Magnetic Separation Method for Intact Lysosomes
一种用于分离完整溶酶体的基于纳米颗粒的磁分离方法   

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Abstract

Lysosome isolation is a preresiquite for identifying lysosomal protein composition by mass spectroscopic analysis, to reveal lysosome functions, and their involvement in some diseases. Magnetic nanoparticle-based fractionation has received great attention for lysosome isolation, owing to its high efficiency, purity, and preservation of lysosomal structures. Understanding the intracellular trafficking of magnetic probes is the key point of this technique, to determine the appropriate time for magnetic isolation of lysosomes, because this parameter changes depending on different cell lines used. The traditional magnetic probes, such as superparamagnetic iron oxide nanoparticles (SPIONs), require surface modification by fluorescent dyes to enable the investigation of their intracellular trafficking, which has some disadvantages, including the possible alternation of their bio-interaction, and the instability of fluorescence properties in the lysosomal environment. To overcome those limitations, we present a protocol that employs magnetic-plasmonic nanoparticles (MPNPs) to investigate intracellular trafficking using their intrinsic imaging capability, followed by quick lysosome isolation using a magnetic column. This protocol can be easily applied to isolate the intact lysosomes of any adherent cell lines.


Graphical abstract:


Keywords: Lysosomes (溶酶体), Nanoparticles (纳米颗粒), Magnetic separation (磁分离), Plasmonic imaging (等离子体成像), Endocytosis (内吞作用), Endolysosomal pathway (内溶酶体途径), Intracellular trafficking (细胞内运输)

Background

Since their discovery by Christian de Duve in the 1950s (De Duve et al., 1955), the role of lysosomes in cellular function has been explored extensively, which led to the change of the view of lysosomes from a static digestive system, to the dynamic regulator of cellular metabolism. As indicated in various studies, lysosomal dysfunctions are found to be linked with the group of metabolic disorders known as lysosomal storage diseases (Mukherjee et al., 2019). Therefore, understanding lysosomal biology in both normal and pathogenic conditions is crucial to figuring out the mechanistic insights of lysosomal activity, to facilitate diagnostic methods, or establish a new therapeutic strategy.


The rapid and efficient isolation of lysosomes is a prerequisite to identify lysosomal protein composition, using proteomic analysis to reveal their involvement in cellular functions or disease progression. So far, several strategies have been developed to isolate lysosomes, including density-gradient centrifugation, immunoaffinity purification, and magnetic nanoparticle-based fractionation. Among these approaches, a nanoparticle-based method that delivers magnetic nanoparticles to the lumen of lysosomes, through an endocytic pathway, followed by a separation process, using a magnetic column, has been proven to be able to isolate lysosomes with the highest yield and purity, while efficiently preserving their integrity (Singh et al., 2020).


The accurate understanding of intracellular trafficking of magnetic nanoparticles is a key step to prevent contamination by other organelles (i.e., endosomes) in the magnetic nanoparticles-based fractionation of lysosomes. Generally, SPIONs are used as magnetic probes, which generally requires employing fluorescent dye-based techniques to monitor their intracellular trafficking. However, it has been suggested that the lysosomal environment could lead to quenching and/or distortion of fluorescence dye signals, which may cause an ensuing effect on the interpretation of the data (Milosevic et al., 2017). In addition, the surface modification of nanoparticles with dye molecules may influence the nano-bio interactions, which results in the alteration of the cellular uptake and intracellular trafficking of nanoparticles (Snipstad et al., 2017; Thomsen et al., 2021). Herein, to further refine the magnetic nanoparticle-based fractionation, the magnetic-plasmonic Ag/FeCo/Ag core/shell/shell nanoparticles (MPNPs) are used as multifunctional probes for lysosome isolation. Owing to their plasmonic properties, the intracellular trafficking of MPNPs can be easily investigated using confocal laser scanning microscopy, to confirm the accumulation of MPNPs in lysosomes, prior to magnetic isolation.


This protocol outlines the optimized procedures for preparation of MPNPs, intracellular trafficking study of MPNPs, and magnetic isolation of lysosomes. The time required for completing magnetic isolation of lysosomes after cell homogenization is within 30 min, which is significantly shorter than that of the density-gradient centrifugation technique. The amount of protein obtained was sufficient for mass spectroscopy, to identify protein composition. More importantly, this protocol was demonstrated to be easily adaptable to other adherent cell lines (Le et al., 2022).

Materials and Reagents

  1. Glass syringe with lock tip 2 mL (Cadence Science, Stock Keeping Unit: 2407)

  2. Glass syringe with lock tip 5 mL (Cadence Science, Stock Keeping Unit: 2417)

  3. Stainless steel 304 syringe needle, noncoring point 2 inch 12G (Sigma-Aldrich, catalog number: Z116947-1EA)

  4. Stainless steel 304 syringe needle, noncoring point 6 inch 20G (Sigma-Aldrich, catalog number: Z102709-1EA)

  5. Centrifuge tube 50 mL (AS One, catalog number: 2-3939-03)

  6. Microtube 1.5 mL (AS One, L-2057)

  7. VIOLAMO 5 mL tube (AS One, catalog number: 2-4118-01)

  8. Centrifuge tube 15 mL (AS One, catalog number: 1-3500-21)

  9. Round cover glass Φ12mm No.1 (Matsunami, catalog number: C012001)

  10. White slide glass edge grinding S1111 (AS One, catalog number: 2-154-01)

  11. Terumo syringe with needle 2.5 mL 23G blue (AS One, catalog number: 1-2044-03)

  12. Parafilm membrane (Amcor, Parafilm M, catalog number: PM996)

  13. CELLect® Fetal bovine serum, 500 mL (FBS; MP Biomedicals, catalog number: 2917354H)

  14. High-purity Ar gas, >99.9999 vol.%

  15. Cobalt (II) acetylacetonate, 97% (Co precusor; Sigma-Aldrich, catalog number: 227129-50G)

  16. Iron (III) acetylacetonate, 99.99% (Fe precusor; Sigma-Aldrich, catalog number: 517003-50G)

  17. Silver nitrate, 99.9999% (Ag precusor; Sigma-Aldrich, catalog number: 204390-10G)

  18. 1,2-hexadecanediol, 90% (Sigma-Aldrich, catalog number: 213748-50G)

  19. Oleylamine, 70% (Sigma-Aldrich, catalog number: O7805-500G), stored at 4°C

  20. Oleic acid, 90% (Sigma-Aldrich, catalog number: 364525-1L), stored at 4°C

  21. Tetraethylene glycol (Sigma-Aldrich, catalog number: 110175-1KG)

  22. Acetone, 99.5% (Kanto Chemical, catalog number: 01026-70)

  23. Hexane, 96% (Kanto Chemical, catalog number: 18041-70)

  24. Chloroform, 99% (Kanto Chemical, catalog number: 07278-70)

  25. Toluene, 99% (Wako Pure Chemical, catalog number: 201-01871)

  26. 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350] (PEG350-DOPE; Avanti, catalog number: 880430O-25MG), stored at −20°C

  27. 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-(glutaryl) (18:1 Glutaryl PE; Avanti, catalog number: 870242C-25MG), stored at -20°C

  28. 2-morpholinoethanesulfonic acid, monohydrate, (MES; Dojindo, catalog number: 341-01622)

  29. N-hydroxysuccinimide (NHS; Thermo Fisher Scientific, catalog number: 24500), stored at 4°C

  30. Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC; Dojindo, catalog number: 346-03632), stored at 4°C

  31. Amino dextran, MW. 10,000 (aDxt; Thermo Fisher Scientific, catalog number: D1860), stored at 4°C

  32. Dulbecco’s phosphate buffer (PBS; Nissui Pharmaceutical, catalog number: 05913), stored at 4°C

  33. Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, catalog number: 08456-36), stored at 4°C

  34. COS-1 cells (available from American Type Culture Collection, catalog number: CRL-1650)

  35. Poly-L-lysine (PLL) solution, 0.01% (Sigma-Aldrich, catalog number: P4832-50ML)

  36. 4%-paraformaldehyde phosphate buffer, 500 mL (PFA; Nacalai Tesque, catalog number: 09154-85), stored at 4°C

  37. Digitonin (Wako Pure Chemical, catalog number: 043-21376), stored at 4°C

  38. Ammonium chloride (NH4Cl, Wako Pure Chemical, catalog number: 015-02991)

  39. Bovine serum albumin (BSA; Sigma-Aldrich, catalog number: A8022-50G), stored at 4°C

  40. Alexa Fluor® 647 mouse anti-human CD107A (AF647@CD107A; BD Biosciences, catalog number: 562622), stored at 4°C

  41. 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, catalog number: D1306), stored at 4°C

  42. VECTASHIELD® Antifade mounting medium (Vector Laboratories, catalog number: H-1700), stored at 4°C

  43. MES buffer (0.1 M, pH ~6) (see Recipes)

  44. PBS buffer (see Recipes)

  45. Digitonin solution (see Recipes)

  46. Ammonium chloride solution (see Recipes)

  47. DAPI staining solution (see Recipes)

  48. Protease inhibitor solution (PIS) (see Recipes)

    Note: The specific storage temperatures are indicated. Otherwise, chemicals are stored at room temperature (RT).

Equipment

  1. Analytical balance (Sartorius, model: ME253P)

  2. Three-neck round bottom flask 50 mL with angled side necks, center joint: ST/NS 29/42, side joints: ST/NS 15/25 (Tokyo Garasu Kikai, catalog number: 371-13-06-01)

  3. Strong magnetic stirrer oval Φ12 × 25 mm (AS One, catalog number: 4-2687-04)

  4. Laboran screw tube bottle 13.5 mL (glass vials; AS One, catalog number: 9-852-06)

  5. Liebig condenser 300 mm, bottom joint: 29/42, top joint: 19/38 (Tokyo Garasu Kikai, catalog number: 330-15-51-14)

  6. Digital high accuracy temperature controller (AS One, TJA-550, catalog number: 1-6124-01)

  7. Mantle heater 50 mL (Tokyo Technological Labo, model: S-05)

  8. High power stirrer (AS One, HPS-100, catalog number: 1-4136-01)

  9. Flowmeter (Kofloc, model: RK1250)

  10. Septum rubber, white, natural, for 18 mm tube (FUJIFILM Wako Pure Chemical, catalog number: 195-11771, Japanese Article Number: 4987481378957)

  11. FisherbrandTM Pasteur pipets (Fisher Scientific, catalog number: 22-063156)

  12. Double element thermocouple WK-Φ3.2×200 (AS One, catalog number: 3-9391-14)

  13. Trap sphere, top and bottom joints: 29/42 (Tokyo Garasu Kikai, catalog number: 330-15-91-07)

  14. Refrigerated centrifuge (Kubota, model: 5910 (with RS-410M rotor))

  15. Ultraviolet-visible absorption spectrophotometer (JASCO, model: V-750)

  16. Two-neck round bottom flask 50 mL with an angled side neck, center joint, and side joints: 14/24

  17. TS one-neck round bottom flask 100 mL,15/25, with the glass stopper (Climbing Co., ltd., CL0070-05-11)

  18. Sonicator (AS One, Ultrasonic Cleaner ASU-6, oscillation frequency: 40 kHz)

  19. High-speed micro centrifuge (Hitachi Koki, model: Himac CF15RXII (with T16A31 rotor))

  20. Ultracentrifuge (Eppendorf Himac Technologies, model: CS100FNX (with S100AT4-2004 rotor))

  21. 37°C and 5% CO2 incubator (ESPEC, model: BNA-111)

  22. Confocal laser scanning microscope (CLSM; Olympus, model: FV1000D)

  23. Cell Lifter (Corning, product number: 3008)

  24. MidiMACS separator starting kits (Miltenyi Biotec, catalog number: 130-042-301)

  25. MS Column (Miltenyi Biotec, catalog number: 130-042-201)

  26. High-speed refrigerated micro centrifuge [Tomy Seiko, model: MDX-310 (with AR015-24 rotor)]

Software

  1. Fiji (NIH/https://imagej.net/software/fiji/), with color clustering and coloc 2 plugins

Procedure

  1. Preparation of MPNPs, by a combination of a polyol, and a one-pot synthesis

    Note: The details on the formation mechanism of MPNPs using this method can be found in Takahashi et al. (2015).

    The glassware for synthesizing MPNPs were shown in Figure 1.



    Figure 1. Glassware for preparation of MPNPs.


    (A) Trap sphere, (B) Three-neck round bottom flask, (C) Liebig type reflux condenser, (D) Glass syringes with needles, (E) Clamp, (F) Oval magnetic stir bar, (G) Pasteur pipette, (H) Glass vials, (I) Septum rubbers, (J) Needle, and (K) Thermocouple.

    1. Weigh 0.1 mmol of silver nitrate, and 1.0 mmol of 1,2-hexadecanediol.

    2. Place an oval magnetic stir bar in the three-neck round bottom flask, and transfer the weighed silver nitrate followed by 1,2-hexadecanediol into the flask. Then, sequentially, add 10 mL of tetraethylene glycol, 10 mmol (3.29 mL) of oleylamine, and 8 mmol (2.55 mL) of oleic acid, using a pipette.

      Note: After removing oleyamine and oleic acid bottles from the refrigerator, place them into a water bath at 35°C until completely melted, then take the required volume using a pipette.

    3. Prepare two 13.5-mL glass vials.

    4. Weigh 0.2 mmol of cobalt (II) acetylacetonate, and 0.2 mmol of iron (III) acetylacetonate. Transfer them to a glass vial labeled as Co and Fe precursors. Then, sequentially, add 2 mL of oleylamine, and 1 mL of toluene.

    5. Weigh 0.1 mmol of silver nitrate, and transfer it to the remaining glass vial labeled as Ag precursor. Then, sequentially, add 1 ml of oleylamine, and 1 mL of toluene.

    6. Seal the caps of the two vials with a parafilm membrane, and place them in a sonicator with High Power Mode, for complete dissolution of all the reagents.

      Note: To dissolve the reagents quickly, the vials could be warmed at approximately 40°C during this process. There is no time limit for this step, but they should be ready before the injection steps.

    7. Prepare two septums, as shown in Figure 2.

      Note: Use the 12G-needle to make a hole, to insert the Pasteur pipette into the rubber septum. Adding some ethanol to the hole makes the insertion easier. Ethanol will evaporate after insertion.



      Figure 2. The septum rubbers prepared for Ar bubbling.

      Later on, the needle is removed from the septum (top) to insert a thermocouple.


    8. Place the three-neck round bottom flask containing the raw reaction materials (prepared in Step A2) on the mantle heater.

    9. Connect the trap sphere to the center neck of the flask, and hold them with a clamp. Then, plug in the condenser tube, and connect the other end of the condenser to a trapper containing liquid paraffin (Figure 3).

      Note: The reflux condenser uses air without running water.



      Figure 3. The illustration (A) and photograph (B) of the experimental setup for preparing MPNPs.


    10. Seal the two remaining open necks using the septums shown in Figure 2.

      Note: The tip of the Pasteur pipette for Ar bubbling should be dipped into reaction solution, but not touch the magnetic stir bar.

    11. Turn on the magnetic stirrer at a speed of 150 rpm, and pump in Ar gas through the Pasteur pipette, at a flow rate of 0.35 L/min. The gas flows out through the 12G-needle. Leave it for 5 min, to complete the replacement of the atmosphere in the flask with Ar gas.

    12. Remove the needle, and insert a thermocouple.

    13. Turn on the temperature controller, and set the temperature to 100°C.

      Note: Due to the high viscosity of the solution at RT, the stirring speed cannot be set immediately at 500 rpm. Therefore, while the temperature increases, increase the stirring speed slowly. At 50°C, the stirring speed could be fully set at 500 rpm. The heating rate of this step is about 12−13°C/min.

    14. From the moment the temperature reaches 100°C, maintain it for 10 min. During this step, the silver seeds are formed.

      Note: Overheating may be observed, in which the temperature is elevated above 100°C. Slightly lowering the heating mantel to reduce its contact with the flask will reduce the temperature.

    15. After 10 min, increase the temperature to 250°C, by setting the temperature controller at 300°C.
      Note: The purpose of this setting is to linearly rise the temperature up to 250°C (Figure 4).



      Figure 4. Temperature profile of the preparation process of MPNPs.


    16. During the temperature increase, once it reaches 170°C, inject the pre-prepared mixture of Fe and Co precursors, using a glass syringe and 20 G-needle.

      Note: The needle is inserted via the septum containing the thermocouple. Inject the solution drop by drop at a fast pace, for a duration of 1 min. The temperature should be kept around 170°C, by slightly lowering the heating mantle to reduce contact with the flask. The heating rate from 100°C to 170°C is about 12°C/min.

    17. Once the temperature reaches 250°C, inject the pre-prepared Ag precursor using a glass syringe and another 20G-needle. Then, immediately reset the temperature of the controller to 230°C. Maintain the reaction for 10 min.

      Note: This is the most important step. The injection of Ag precursor is normally done in about 20 s. It should not be less than 10 s, or longer than 30 s. After the injection, slightly lower the heating mantle to reduce contact with the flask to avoid overheating. We confirmed that reaction time can be prolonged to 15 min, but the quality of the MPNPs was not influenced. The heating rate from 170°C to 250°C is about 9°C/min.

    18. Set the temperature controller to 0°C to stop heating, and remove the mantle heater. Wait for the reaction system to cool off naturally, while continuing stirring and Ar bubbling. At this time, some of the synthesized particles will be attached to the magnetic stirring bar, but they will be redispersed again during the cooling process.

    19. Once the temperature of the reaction solution is less than 70°C, stop Ar gas flow, and turn off the magnetic stirrer.

    20. Carefully disassemble the setup, and use a pipette to transfer the reaction solution from the three-neck flask to two 50-mL centrifuge tubes evenly.

    21. Add acetone, to fill the tube to 45 mL. Then, perform centrifugation using a Kubota 5910 at 4,640 × g and RT for 5 min.

    22. Discard all supernatant, and add 400 µL of hexane to each tube, for redispersion of MPNPs.

    23. Use a micropipette to transfer 200 µL of redispersed MPNP solution to two other 50-mL tubes. Subsequently, fill with acetone up to 45 mL in total, and perform centrifugation using Kubota 5910 at 4,640 × g and RT for 5 min.

    24. Discard the supernatant, and redisperse the obtained MPNPs in 3 mL of chloroform. Determine the concentration of MPNP dispersion through the absorption spectrum, using an ultraviolet-visible absorption spectrophotometer.

      Note: The concentration of MPNPs was determined using a calibration curve of y = 0.024x, where y was the absorption peak value of localized surface plasmon resonance of MPNPs, and x was the concentration of MPNPs (µg/mL).

    25. Store obtained MPNPs in chloroform at 4°C, in a glass vial with closed-top cap. Seal it with Parafilm.


  2. Encapsulation of MPNPs in PEGylated phospholipids

    1. Prepare a 50-mL two-neck round bottom flask (Figure 5A).

    2. Pour 3 mL of MPNPs dispersed in chloroform at a concentration of 1 mg/mL into the flask. Subsequently, add 1,350 µL of 18:1 glutaryl PE (5.5 mM) in chloroform, and 900 µL of PEG350-DOPE (5.5 mM) in chloroform to the dispersion.

    3. Seal using septum rubbers, place the septum containing the Pasteur pipette in the center neck, and the septum containing the needle in the angled neck.

      Note: The tip of the Pasteur pipette should not dip in the dispersion.

    4. Pump in Ar gas at a rate of 0.5 L/min, to completely evaporate the chloroform (Figure 5B).

      Note: In this step, the inert gas could be either Ar or N2.



      Figure 5. The glassware (A) and the experimental setup (B) for encapsulation of MPNPs in PEGylated phospholipids.


    5. Add 1.5 mL of deionized water to redisperse the obtained precipitation. Collect the dispersion into two 1.5-mL centrifuge tubes.

    6. Centrifuge at 1,400 × g and RT for 5 min, using the Hitachi CF15RXII high-speed micro centrifuge, to eliminate big aggregated particles.

    7. Carefully transfer the supernatant from the centrifugated tubes into two new 1.5-mL tubes. Then, centrifuge at 86,600 × g and 4°C for 10 min, using the CS100FNX ultracentrifuge, to remove empty micelles.

      Note: In this step, if the MPNPs were not completely collected, increase the centrifugation speed up to 100,000 × g.

    8. Discard the supernatant, and redisperse the obtained particles in 1 mL of deionized water.

    9. Determine the concentration of phospholipid encapsulated MPNPs from the absorption spectrum.


  3. Conjugation of aDxt using EDC coupling reaction

    1. Add 20 mL of MES buffer (pH ~6) to a 100-mL one-neck round bottom flask, containing a magnetic stirrer.

    2. Set the magnetic stirrer to 600 rpm.

    3. Add 1 mL of phospholipid encapsulated MPNPs dispersion (1 mg/mL) into the flask. Then, sequentially add 125 µL of EDC (200 mM) in deionized water, and 250 µL of NHS (200 mM) in MES buffer. Leave it at RT for 30 min.

      Note: After taking them out from the refrigerator, equilibrate the EDC and NHS to RT before use.

    4. Transfer the obtained reaction mixture into 20 × 1.5-mL tubes.

    5. Centrifuge at 86,600 × g and 4°C for 10 min, using the CS100FNX, and carefully remove the supernatant.

    6. Use a micropipette to collect, and redisperse the obtained particles in 1 mL of PBS.

    7. Prepare a 100-mL one-neck round-bottom flask, containing 19 mL of PBS.

    8. Add 50 mg of aDxt into the PBS solution in the flask (prepared in Step C7), and wait for it to completely dissolve under magnetic stirring.

    9. Add the PBS dispersion of MPNPs (prepared in Step C6) into the PBS solution of aDxt (prepared in Step C8). Then, maintain the reaction at RT for 90 min.

    10. Transfer the obtained mixture to 20 × 1.5-mL tubes. Then, perform centrifugation at 60,000 × g and 4°C for 10 min, using the CS100FNX. Collect, and redisperse the obtained particles in 1 mL of PBS.

    11. Determine the concentration of aDxt-conjugated MPNPs (aDxt-MPNPs) in the dispersion, using the absorption spectrum.


  4. Pulse-chase experiments for studying intracellular trafficking of MPNPs

    Notes:

    1. In a pulse-chase experiment, the pulse is the incubation of aDxt-MPNPs with COS-1 cells for a certain period (tload). After the loading step, the excess amount of aDxt-MPNPs that are not incorporated in the cells is removed. Cells are further incubated in a fresh culture medium for a given period, tchase. The purpose of this experiment is to find the optimal tchase, for aDxt-MPNPs to reach lysosomal compartments through the endolysosomal pathway.

    2. These experiments are performed on a clean bench under sterile conditions.

    1. Place 10–20 sterilized round cover glasses into a 10-cm culture dish.

    2. Add 5 mL of 0.01% PLL solution into the 10-cm dish, and dip the round cover glasses in PLL solution at RT for 5 min, using a tweezer.

    3. Remove the PLL solution, cover with aluminum foil, with the foil partially opened, and naturally dry overnight on a clean bench.

    4. The next day, wash the PLL coated cover glasses three times, using 5 mL of PBS buffer.

    5. Place the four cover glasses in each well of a 24-well plate using a tweezer, which correspond to different tchase values of 1 h, 2 h, 4 h, and 7 h.

      Note: The number of cover glasses increases according to the number of investigated tchase. In the case of COS-1 cells, the maximum tchase was performed at 7 h. However, it should be noted that the length of tchase depends on different cell lines. Therefore, the incubation time of this experiment could be customized easily.

    6. Seed 20,000 COS-1 cells/well and incubate overnight in DMEM (+10% FBS) at 37°C under 5% CO2.

    7. Check the health and confluency of cells, under a bright-field microscope (Keyence, model: BZ-X810) in advance.

    8. The next day, remove the culture medium, and wash cells with 500 µL of PBS at RT.

    9. For cell starvation, add 0.5 mL of pre-warmed DMEM without FBS, and incubate for 30 min at 37°C under 5% CO2.

    10. Approximately 10 min before finishing the starvation process, add MPNPs to DMEM (+10% FBS), to prepare a dispersion of MPNPs with the concentration of 100 µg/mL.

    11. Immediately affter starvation, replace DMEM without FBS with 500 µL of MPNPs dispersion in DMEM (+10% FBS) (prepared in Step D10), and incubate for tload = 1 h at 37°C under 5% CO2.

    12. After 1 h incubation, remove the dispersion, and wash with 500 µL of PBS once. Then, add 500 µL of pre-warmed DMEM (+10% FBS), and incubate for tchase: 0 h, 2 h, 4 h, and 7 h at 37°C under 5% CO2.

      Note: The tchase would be varied in different cell lines. Therefore, the incubation period could be customized appropriately.

    13. After completing the tchase, wash with 500 µL of PBS three times, and add 500 µL of 4% PFA at RT to each well for 15 min.

      Note: Since PFA is a toxic chemical, personnel must wear a lab coat and chemically protective gloves. This step should be performed in a clean bench equipped with a ventilation system and a protective sash. Additionally, keep PFA solution away from flame or heat sources. The PFA should be properly disposed of as hazardous waste. After fixation, the following steps could be performed outside the clean bench. The sterile conditions are not required.

    14. Wash with 500 µL of PBS three times, add 500 µL of 50 µg/mL digitonin-PBS for permeabilization to each well, and wait for 5 min.

    15. Wash with 500 µL of PBS three times, add 500 µL of 50 mM NH4Cl-PBS to each well, and wait for 10 min.

    16. Wash with 500 µL of PBS three times, and perform blocking by adding 500 µL of 3 wt% BSA-PBS to each well, and waiting for 30 min.

    17. For each well, add 500 µL of 3 wt% BSA-PBS containing 2 µL of AF647@CD107A for staining the lysosomes, and 0.25 µL of 100 µg/mL DAPI for staining the nuclei. Wait at RT for 1 h, or keep it at 4°C overnight.

    18. Wash with 500 µL of PBS three times. For each washing step, wait for 5 min after adding PBS.

    19. Add a drop of antifade mounting medium onto a white slide glass edge grinding, carefully take the cover glass using tweezers, and place it onto the glass substrate for observation with the cell-facing surface in contact with the mounting medium. Ensure there are no bubbles and remove extra fluid if necessary.

    20. Leave it in a dark place for several hours until it is completely dry. Then, observe the samples using a CLSM.


  5. Observation of MPNPs-loaded cells under CLSM

    1. Select 405, 473, and 635 nm lasers for the excitation of DAPI, aDxt-MPNPs, and AF647, respectively.

    2. For DAPI dye, select the barrier filter (BA) 435–455 nm.

    3. For plasmonic scattering signal of aDxt-MPNPs, select no barrier filter, as, unlike the fluorescent dye, the scattering signal from aDxt-MPNPs has the same wavelength as the laser wavelength.

    4. For AF647 dye, select BA 655–755 nm.

    5. Capture CLSM images of more than five different randomly-selected regions. Record DAPI signal separately to plasmonic scattering signal and AF647 fluorescence (Figure 6).

    6. Perform colocalization analysis of aDxt-MPNPs and lysosomes, by determining the threshold Manders’ colocalization coefficient (Rt), using ImageJ software.



      Figure 6. CLSM images of aDxt-MPNP-loaded COS-1 cells at different tchase values of 0 h, 2 h, 4 h, and 7 h, captured by CLSM (scale bar: 20 µm).

      Nuclei (blue) and lysosomes (red) are stained by DAPI and AF647, respectively, as described in the text. aDxt-MPNPs were observed by plasmonic scattering signals. The merged images were obtained using ImageJ. Adapted with permission from Le et al. (2022). Copyright 2022 American Chemical Society.


  6. Accumulation of aDxt-MPNPs to lysosomes, homogenization, and magnetic isolation of lysosomes

    1. Seed 2 × 106 COS-1 cells/dish for two 10-cm dishes, and incubate in DMEM (+10% FBS) for 24 h.

    2. Check the health and confluency of cells under a bright-field microscope (Keyence, model: BZ-X810) in advance.

      Note: The health of COS-1 cells was checked by confirming their adherent status on a cell dish, using bright-field microscopy. In addition, the possibility of contamination was also checked at the same time. The confluency of cells was estimated to be less than 80% in this particular experiment. However, it would change depending on the cell type. The desired confluency of cells would be ranging from 70% to 80%. If cells are well adhered on the dish without being contaminated, and with around 70-80% confluency, one can go to the next step.

    3. For cell starvation, add 5 mL of pre-warmed DMEM without FBS to each cell dishes, and incubate at 37°C under 5% CO2 for 30 min.

    4. About 10 min before finishing the starvation process, add MPNPs to DMEM (+10% FBS), to prepare a 10-mL of dispersion of MPNPs, with a concentration of 100 µg/mL.

    5. Immediately after starvation, replace DMEM without FBS with 5 mL of MPNPs dispersion in DMEM (+10% FBS) (prepared in Step F4) to each cell dish, and incubate at 37ºC under 5% CO2 for 8 h.

      Note: In this study, we chose the tload = 8 h for loading. tload strongly affects the isolation yield of lysosomes. This parameter could be prolonged depending on the cytotoxicity of MPNPs to the cells.

    6. Discard the aDxt-MPNPs containing medium, and wash with pre-warmed PBS once.

    7. Add 5 mL of DMEM (+10% FBS), and incubate further for tchase = 7 h. The optimal tchase has been already determined in the pulse-chase experiment section. Depending on the cell type, this parameter may vary.

    8. Place necessary equipment, including the magnetic column, MidiMACS separator, 2.5-mL syringe with 23G-needle (Terumo syringe with needle 2.5 mL 23G blue), and 5-mL tubes, into the cold room, where the temperature is maintained at 4°C, for at least 30 minutes before the pulse-chase experiment, to equilibrate the temperature. If a cold room is not available, use an ice-box to store the equipment instead.

    9. Discard the medium, and wash the cells with PBS.

    10. Add 1.5 mL of cold PBS to each culture dish, and place them on ice.

    11. Scrape off the cells using a Cell Lifter, and transfer them from both culture dishes to a 15-mL centrifuge tube. Centrifuge at 190 × g and 4°C for 4 min, using the Kubota 5910 with a ST-720 swinging bucker rotor.

      Note: In this step, the amount of particle uptake could be qualitatively evaluated via the color of the cell pellet (Figure 7). The darker the color, the higher number of particles internalized. If the cell color is still white, it means a very low uptake efficiency. The isolation of lysosomes may fail.

    12. Discard the supernatant, and add 1 mL of ice-cold PIS to re-suspend the cell pellet. Then, transfer to a 5-mL tube, and keep in an icebox.

      Note: After this step, the experiments are continued in a low-temperature room, where the temperature is maintained at 4°C.



      Figure 7. A photograph of aDxt-MPNPs loaded COS-1 cells with tload = 8 h and tchase = 7 h.


    13. Use a 2.5-mL syringe with a 23G-needle, and repeatedly (15 passages) pass the cell suspension through the syringe, to homogenize the cells.

      Note: The optimal number of passages must be determined experimentally (Figure 8). The low homogenization efficiency could obviously affect the yield of lysosome isolation. In contrast, homogenization efficiency enhanced by increasing the number of passages may also lead to lysosomes being broken. Therefore, in this study, a small portion of unbroken cells or large cell fragments is left over.



      Figure 8. The bright-field image of COS-1 cells under phase contrast mode.

      Before (A) and after (B) homogenization using a syringe with a 23G-needle (15 passages). Before homogenization, cells can clearly be seen as a high density of dark areas encircled by bright halos. After homogenization, the number of cells is reduced, and the cell mixture becomes a slurry, due to the breaking of the cell membranes. Consequently, the number of bright halos decreases significantly. A small portion of either unbroken cells or large cell fragments in the slurry can still be observed. Scale bar, 100 µm.


    14. Place an MS Column in a MidiMACS separator.

      Note: Another type of MACS® Column, such as LS Column, could also be used in this experiment.

    15. Equilibrate the MS Column, by adding 1 mL of PIS. Discard the flow-through.

    16. Transfer the cell lysate (prepared in Step F13) to the MS Column, using the micropipette, and allow the cell lysate to pass through the column. The magnetic fraction will be trapped inside the column, while the nonmagnetic fraction will pass through the column.

    17. Discard the flow through. Wash the column using 1 mL of PIS twice, to further eliminate unbound materials.

    18. Remove the MS Column from the MidiMACS separator.

    19. Add 0.5 mL of PIS, and insert the plunger to collect the magnetic fraction containing lysosomes in a 1.5-mL microtube. Repeat this step once again.

    20. Centrifuge the obtained suspension at 5,000 × g and 4°C for 10 min, using an MDX-310 system.

      Note: This step is to remove the remaining soluble proteins in the isolated fraction.

    21. Discard the supernatant, and redisperse the obtained lysosome pellet in 100 µL of PIS.

      Note: If the isolated lysosome fraction is subjected to proteome analysis, re-suspend the pellet in 50 mM triethylammonium bicarbonate.

Data analysis

The colocalization analysis of aDxt-MPNPs and lysosomes is performed using ImageJ. First, open the image, then choose “Plugin” → “Segmentation” → “Color Clustering”. Afterward, in the new window, in the “Channel” section, choose the appropriate color for the image. For this specific case, the illustrated colors of aDxt-MPNPs and lysosomes are green and red, respectively. Next, press “Run”, and then choose “Show result” to obtain the segmented image. Note that only one image of either aDxt-MPNPs or lysosomes can be processed at once (Figure 9). After obtaining segmented images, the Rt represents the percentage of lysosomes overlapped with aDxt-MPNPs. To open color clustered images of aDxt-MPNPs and lysosomes, select “Analyze” → “Colocalization” → “Coloc 2”. In the new window, choose the image of aDxt-MPNPs for channel 1, and the image of lysosomes for channel 2, then check the Manders’ correlation box, and press OK (Figure 10). Repeat this step for five pairs of images from each experimental condition.



Figure 9. Segmentation of CLSM images using color clustering.

(A) Select “Plugin” > “Segmentation” > “Color Clustering”. (B) For an aDxt-MPNP image, the illustrated color of nanoparticles is green, therefore, select green (in the channel section), and click “Run”. Repeat this step for the CLSM image of stained lysosomes, and select the appropriate color for segmentation accordingly.



Figure 10. The process to determine the Rt using Coloc 2 plugin in ImageJ software.

(A) Open two segmented images in the previous steps, select “Analyze” > “Colocalization” > “Coloc 2”. (B) In the new window, select images for channel 1 and channel 2. Then, check the Manders’ correlation box, and click OK.


After completing the image analysis, a graph of Rt-versus-incubation time can be constructed (Figure 11A). The incubation time is the sum of tload and tchase. As aDxt-MPNPs are transported to lysosomes, the Rt value increases. However, due to the limited spatial resolution of the CLSM image, the Rt value is saturated. From this graph, the value tchase can be determined. Normally, the tchase is chosen after one time when Rt reaches a plateau. The reasoning behind this is that, in the endolysosomal pathway, late-endosomes are fused with lysosomes, which could also result in the high colocalization of aDxt-MPNPs with lysosomes. However, it is recommended that, after obtaining the isolated lysosome fraction, the level of late endosomes should be evaluated using Western blot analysis (Figure 11B). If the late endosome still exists, a further prolonged tchase is necessary.



Figure 11. Time-lapse colocalization of aDxt-MPNPs with lysosomes and Western blot of cell lysate, PS, and NS fractions.

(A) The graph of Rt-versus-incubation time. The accumulation of aDxt-MPNPs in lysosomes is indicated by the increase of Rt over time. (B) A western blot of the isolated lysosome fraction. PS: positive selection (magnetic fraction); NS: negative selection (nonmagnetic fraction); GAPDH: glyceraldehyde-3-phosphate dehydrogenase (cytosolic protein as a control); LAMP2: lysosomal associated membrane protein 2 (lysosome marker), Rab7: late endosome marker protein. Adapted with permission from Le et al. (2022). Copyright 2022 American Chemical Society.

Recipes

  1. MES buffer (0.1 M, pH ~6)

    Dissolve 3.90 g MES in 180 mL of deionized water. Monitor the pH of the solution using a pH meter, then take 10 N sodium hydroxide aqueous solution using a micropipette, to adjust the pH of the solution to approximately 6. Then, add water up to 200 mL. Sterilize the solution by filtration through a 0.2-µm filter before use. Store in a dark colored bottle.

  2. PBS buffer

    Dissolve 9.6 g of PBS in 1 L of deionized water. The solution should be sterilized by an autoclave before use. Store the solution at 4°C.

  3. Digitonin solution

    Dissolve 25 mg of digitonin in 500 µL of dimethyl sulfoxide (DMSO). Divide the solution into microtubes, at 15 µL/tube. Store the solution at -20°C. It is diluted with PBS, for permeabilization.

  4. Ammonium chloride solution

    Dissolve 0.160 g NH4Cl in 60 mL of PBS buffer. Store the solution at 4°C.

  5. DAPI staining solution

    From the commercial product, prepare the DAPI stock solution with a concentration of 100 µg/mL, store in the refrigerator at 4°C. For nucleus staining, dilute the stock solution 2000 times further.

  6. Protease inhibitor solution (PIS)

    1. Prepare 20 mL of PBS in a 50-mL tube.

    2. Add 20 μL of 0.1 M phenylmethylsulfonyl fluoride and 100 µL of protease inhibitor cocktail to the tube. The dilution factor is about 1000× and 200× for phenylmethylsulfonyl fluoride and protease inhibitor cocktail, respectively.

    3. Disperse the solution homogeneously using a vortex. Then, keep the solution in an ice box.

    Note: This solution should be prepared at the time of use. Long-term storage is not recommended. The composition of the inhibitor cocktail is: 0.1 mg/mL leupeptin hemisulfate monohydrate; 0.14 mg/mL pepstatin A; 14 mg/mL N-p-tosyl-L-phenylalanine chloromethyl ketone; 15 mg/mL Nα-p-tosyl-L-arginine methyl ester hydrochloride; 0.4 mg/mL aprotinin; 32 mg/mL benzamidine dissolved in DMSO. The inhibitor cocktail can be prepared in advance and stored in small tubes at -20°C.

Acknowledgments

This protocol is derived from the original research paper, Le et al. “Quick and Mild Isolation of Intact Lysosomes Using Magnetic−Plasmonic Hybrid Nanoparticles” ACS Nano 2022 Jan 3; 16(1): 885–896. doi: 10.1021/acsnano.1c08474 (Le et al., 2022). This work was partly funded by the Grant-in-Aid for Young Scientists from the Japan Society for the Promotion of Science (grant no. 21K14506) to M.T.

Competing interests

The authors declare no competing interests.

Ethics

No human or vertebrate animal subjects are used in this study.

References

  1. De Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955). Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J 60(4): 604-617.
  2. Le, T. S., Takahashi, M., Isozumi, N., Miyazato, A., Hiratsuka, Y., Matsumura, K., Taguchi, T. and Maenosono, S. (2022). Quick and Mild Isolation of Intact Lysosomes Using Magnetic–Plasmonic Hybrid Nanoparticles. ACS Nano 16(1): 885-896.
  3. Milosevic, A. M., Rodriguez-Lorenzo, L., Balog, S., Monnier, C. A., Petri-Fink, A. and Rothen-Rutishauser, B. (2017). Assessing the Stability of Fluorescently Encoded Nanoparticles in Lysosomes by Using Complementary Methods. Angew Chem Int Ed Engl 56(43): 13382-13386.
  4. Mukherjee, A. B., Appu, A. P., Sadhukhan, T., Casey, S., Mondal, A., Zhang, Z. and Bagh, M. B. (2019). Emerging new roles of the lysosome and neuronal ceroid lipofuscinoses. Mol Neurodegener 14(1): 4.
  5. Singh, J., Kaade, E., Muntel, J., Bruderer, R., Reiter, L., Thelen, M. and Winter, D. (2020). Systematic Comparison of Strategies for the Enrichment of Lysosomes by Data Independent Acquisition. J Proteome Res 19(1): 371-381.
  6. Snipstad, S., Hak, S., Baghirov, H., Sulheim, E., Morch, Y., Lelu, S., von Haartman, E., Back, M., Nilsson, K. P. R., Klymchenko, A. S., et al. (2017). Labeling nanoparticles: Dye leakage and altered cellular uptake. Cytometry A 91(8): 760-766.
  7. Takahashi, M., Higashimine, K., Mohan, P., Mott, D. M. and Maenosono, S. (2015). Formation mechanism of magnetic–plasmonic Ag@FeCo@Ag core–shell–shell nanoparticles: fact is more interesting than fiction. CrystEngComm 17(36): 6923-6929.
  8. Thomsen, T., Ayoub, A. B., Psaltis, D. and Klok, H. A. (2021). Fluorescence-Based and Fluorescent Label-Free Characterization of Polymer Nanoparticle Decorated T Cells. Biomacromolecules 22(1): 190-200.

简介

[摘要] 溶酶体分离是通过质谱分析鉴定溶酶体蛋白质组成、揭示溶酶体功能及其与某些疾病有关的先决条件。基于磁性纳米颗粒的分级分离因其高效率、纯度和溶酶体结构的保存而在溶酶体分离方面受到了极大的关注。了解磁性探针的细胞内运输是该技术的关键点,以确定溶酶体磁分离的适当时间,因为该参数根据使用的不同细胞系而变化。传统的磁性探针,如超顺磁性氧化铁纳米粒子(SPIONs),需要通过荧光染料进行表面修饰,以研究它们的细胞内运输,这有一些缺点,包括它们的生物相互作用可能发生变化,以及荧光的不稳定性溶酶体环境中的特性。为了克服这些限制,我们提出了一个协议,该协议采用磁性等离子体纳米粒子 (MPNP) 来利用其固有的成像能力研究细胞内贩运,然后使用磁柱进行快速溶酶体分离。该协议可以很容易地用于分离任何贴壁细胞系的完整溶酶体。

图形概要:


[背景] 自 1950 年代 Christian de Duve 发现溶酶体 (De Duve et al. , 1955) 以来,人们对溶酶体在细胞功能中的作用进行了广泛的探索,这导致溶酶体的观点从静态消化系统转变为细胞代谢的动态调节剂。如各种研究所示,溶酶体功能障碍被发现与称为溶酶体贮积病的一组代谢紊乱有关(Mukherjee等人,2019 年)。因此,了解正常和致病条件下的溶酶体生物学对于弄清溶酶体活性的机制见解、促进诊断方法或建立新的治疗策略至关重要。
溶酶体的快速有效分离是识别溶酶体蛋白质组成的先决条件,使用蛋白质组学分析揭示它们参与细胞功能或疾病进展。到目前为止,已经开发了几种分离溶酶体的策略,包括密度梯度离心、免疫亲和纯化和基于磁性纳米颗粒的分级分离。在这些方法中,一种基于纳米颗粒的方法通过内吞途径将磁性纳米颗粒输送到溶酶体的内腔,然后使用磁柱进行分离过程,已被证明能够以最高的产率和纯度分离溶酶体,同时有效地保持其完整性(Singh等人,2020)。
准确了解磁性纳米粒子的细胞内运输是防止在基于磁性纳米粒子的溶酶体分级分离中被其他细胞器(即内体)污染的关键步骤。通常,SPION 用作磁性探针,这通常需要采用基于荧光染料的技术来监测它们的细胞内运输。然而,有人提出溶酶体环境可能导致荧光染料信号的猝灭和/或失真,这可能会对数据的解释产生影响(Milosevic等人,2017 年)。此外,用染料分子对纳米颗粒进行表面修饰可能会影响纳米生物相互作用,从而导致纳米颗粒的细胞摄取和细胞内运输发生改变(Snipstad等人,2017 年;Thomsen等人,2021 年)。在此,为了进一步完善基于磁性纳米颗粒的分级分离,磁性等离子体 Ag/FeCo/Ag 核/壳/壳纳米颗粒 (MPNPs) 被用作溶酶体分离的多功能探针。由于它们的等离子体特性,可以使用共聚焦激光扫描显微镜轻松研究 MPNPs 的细胞内运输,以在磁分离之前确认 MPNPs 在溶酶体中的积累。
该协议概述了 MPNPs 制备、MPNPs 细胞内贩运研究和溶酶体磁分离的优化程序。细胞匀浆后完成溶酶体磁分离所需时间在30 min内,明显短于密度梯度离心技术。获得的蛋白质量足以用于质谱分析,以确定蛋白质组成。更重要的是,该方案被证明可以轻松适应其他贴壁细胞系(Le等人,2022)。

关键字:溶酶体, 纳米颗粒, 磁分离, 等离子体成像, 内吞作用, 内溶酶体途径, 细胞内运输



材料和试剂


1.带锁头的玻璃注射器 2 mL(Cadence Science, S库存单位:2407)
2.带锁头的玻璃注射器 5 mL(Cadence Science, S库存单位:2417)
3.不锈钢304注射器针头,非取芯点2英寸12G(Sigma-Aldrich,目录号: Z116947-1EA)
4.不锈钢304注射器针头,非取芯点6英寸20G(Sigma-Aldrich,目录号: Z102709-1EA)
5.离心管50 mL(AS One,目录号: 2-3939-03)
6.微量管 1.5 mL(AS One,L-2057)
7.VIOLAMO 5 mL管(AS One,目录号: 2-4118-01)
8.离心管15 mL(AS One,目录号: 1-3500-21)
9.圆形盖玻片Φ12mm No.1(Matsunami,目录号: C012001 )
10.白色载玻片玻璃磨边 S1111(AS One,目录号: 2-154-01)
11.带针头的 Terumo 注射器 2.5 mL 23G 蓝色(AS One,目录号: 1-2044-03)
12.Parafilm膜(Amcor,Parafilm M,目录号:PM996)
13.CELLect ®胎牛血清,500 mL(FBS;MP Biomedicals,目录号: 2917354H)
14.高纯氩气,>99.9999 vol.%
15.钴(II)乙酰丙酮化物,97%(Co前体;Sigma-Aldrich,目录号:227129-50G)
16.铁(III)乙酰丙酮化物,99.99%(Fe前体;Sigma-Aldrich,目录号:517003-50G)
17.硝酸银,99.9999%(Ag前体;Sigma-Aldrich,目录号: 204390-10G)
18.1,2-十六烷二醇,90% (Sigma-Aldrich,目录号:213748-50G)
19.油胺,70% (Sigma-Aldrich,目录号:O7805-500G),储存于 4°C
20.油酸,90% (Sigma-Aldrich,目录号:364525-1L),储存于 4°C
21.四甘醇(Sigma-Aldrich,目录号:110175-1KG)
22.丙酮,99.5%(Kanto Chemical,目录号: 01026-70)
23.己烷,96%(Kanto Chemical,目录号: 18041-70)
24.氯仿,99%(Kanto Chemical,目录号: 07278-70)
25.甲苯,99%(Wako Pure Chemical,目录号: 201-01871)
26.1,2-二油酰基-sn-甘油-3-磷酸乙醇胺-N- [甲氧基(聚乙二醇)-350](PEG350-DOPE;Avanti, 目录编号: 880430O-25MG ),储存于 − 20°C
27.1,2-二油酰-sn-甘油-3-磷酸乙醇胺-N- (戊二酰)(18:1戊二酰PE;Avanti,目录号: 870242C-25MG),储存于-20°C
28.2-吗啉代乙磺酸,一水合物,(MES;Dojindo,目录号: 341-01622)
29.N-羟基琥珀酰亚胺(NHS;Thermo Fisher Scientific,目录号: 24500),储存于 4°C
30.乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC;Dojindo,目录号: 346-03632),储存在 4°C
31.氨基葡聚糖,MW。 10,000(aDxt;Thermo Fisher Scientific,目录号: D1860),储存于 4°C
32.Dulbecco 磷酸盐缓冲液(PBS;Nissui Pharmaceutical,目录号:05913),储存于 4°C 
33.Dulbecco 改良 Eagle 培养基(DMEM;Nacalai Tesque, 目录号: 08456-36),储存在 4°C
34.COS-1 细胞(可从美国典型培养物保藏中心获得,目录号: CRL-1650 )
35.-L-赖氨酸(PLL)溶液,0.01%(Sigma-Aldrich,目录号: P4832-50ML )
36.4%-多聚甲醛磷酸盐缓冲液,500 mL(PFA;Nacalai Tesque,目录号: 09154-85),储存于 4°C
37.Digitonin(Wako Pure Chemical,目录号: 043-21376),储存于 4°C 
38.氯化铵(NH 4 Cl,Wako Pure Chemical,目录号: 015-02991)
39.牛血清白蛋白(BSA;Sigma-Aldrich,目录号: A8022-50G),储存于 4°C
40.Alexa Fluor ® 647 小鼠抗人 CD107A(AF647@CD107A;BD Biosciences,目录号: 562622),储存于 4°C
41.4 ',6-二脒基-2-苯基吲哚(DAPI;Thermo Fisher Scientific,目录号: D1306),储存于 4°C
42.VECTASHIELD ® Antifade 封固剂(Vector Laboratories,目录号: H-1700),储存于4°C
43.MES 缓冲液(0.1 M,pH ~6)(见配方)
44.PBS 缓冲液(见配方)
45.洋地黄皂苷溶液(见配方)
46.氯化铵溶液(见配方)
47.DAPI 染色溶液(见配方)
48.蛋白酶抑制剂溶液 (PIS) (见配方)
注:标明了具体的储存温度。否则,化学品将在室温 (RT) 下储存。


设备


1.分析天平(赛多利斯,型号:ME253P)
2.三颈圆底烧瓶 50 mL,带斜颈, 中心接头:ST/NS 29/42,侧接头:ST/NS 15/25(Tokyo Garasu Kikai,目录号: 371-13-06-01)
3.强力磁力搅拌器椭圆形Φ 12 × 25 mm(AS One,目录号: 4-2687-04)
4.Laboran螺旋管瓶13.5 mL(玻璃小瓶;AS One,目录号: 9-852-06)
5.Liebig冷凝器300 mm,底部接头:29/42,顶部接头:19/38(Tokyo Garasu Kikai,目录号: 330-15-51-14) 
6.数字高精度温度控制器(AS One,TJA-550,目录号: 1-6124-01)
7.地幔加热器 50 mL(Tokyo Technological Labo,型号: S-05 )
8.大功率搅拌器(AS One,HPS-100,目录号: 1-4136-01 )
9.流量计(Kofloc,型号:RK1250)
10.隔垫橡胶,白色,天然,用于 18 mm 管(FUJIFILM Wako Pure Chemical,目录号: 195-11771,日本商品编号:4987481378957)
11.Fisherbrand TM巴斯德吸管(Fisher Scientific,目录号: 22-063156)
12.双元件热电偶 WK- Φ 3.2×200(AS One,目录号: 3-9391-14)
13.陷阱球体,顶部和底部接头:29/42(Tokyo Garasu Kikai,目录号: 330-15-91-07)
14.冷冻离心机(Kubota,型号:5910(带 RS-410M 转子))
15.紫外-可见吸收分光光度计(JASCO,型号:V-750)
16.双颈圆底烧瓶 50 mL,带有倾斜的侧颈、中心接头和侧接头:14/24
17.TS 单颈圆底烧瓶 100 mL,15/25,带玻璃塞(Climbing Co., ltd., CL0070-05-11)
18.Sonicator (AS One,超声波清洁器 ASU-6,振荡频率:40 kHz)
19.高速微型离心机(日立工机,型号:Himac CF15RXII(配T16A31转子))
20.超速离心机(Eppendorf Himac Technologies,型号:CS100FNX(带 S100AT4-2004 转子))
21.37 °C和 5% CO 2培养箱(ESPEC,型号:BNA-111)
22.共焦激光扫描显微镜(CLSM;奥林巴斯,型号: FV1000D)
23.Cell Lifter(康宁,产品编号:3008)
24.MidiMACS 分离器起始试剂盒(Miltenyi Biotec,目录号: 130-042-301)
25.MS 柱(Miltenyi Biotec,目录号: 130-042-201)
26.高速 冷藏微型离心机 [Tomy Seiko,型号:MDX-310(配 AR015-24 转子)]


软件


1.斐济(NIH/ https://imagej.net/software/fiji/ ),带有颜色聚类和 coloc 2 插件


程序


A.通过多元醇和一锅法合成制备 MPNPs
注:使用该方法形成 MPNPs 的详细信息可参见 Takahashi 等人。 (2015 年)。
用于合成 MPNPs 的玻璃器皿如图 1 所示。




图1。 用于制备 MPNP 的玻璃器皿。
(A) 捕集球,(B) 三颈圆底烧瓶,(C)李比希型回流冷凝器,(D) 带针头的玻璃注射器,(E) 夹子,(F) 椭圆形磁力搅拌棒,(G) 巴斯德吸管, (H) 玻璃小瓶, (I) 隔垫橡胶, (J) 针和 (K) 热电偶。


1.称取 0.1 mmol 硝酸银和 1.0 mmol 1,2-十六烷二醇。
2.在三颈圆底烧瓶中放置一个椭圆形磁力搅拌棒,然后将称重的硝酸银和 1,2-十六烷二醇转移到烧瓶中。然后,依次使用移液器加入10 mL 的四甘醇、10 mmol(3.29 mL)的油胺和 8 mmol(2.55 mL)的油酸。
注意:从冰箱中取出油胺和油酸瓶后,将它们放入 35°C 的水浴中直至完全融化,然后使用移液器吸取所需体积。
3.准备两个 13.5 mL 玻璃小瓶。
4.称重 0.2 毫摩尔的钴 (II) 乙酰丙酮化物和 0.2 毫摩尔的铁 (III) 乙酰丙酮化物。将它们转移到标记为 Co 和 Fe 前体的玻璃小瓶中。然后,依次加入 2 mL 油胺和 1 mL 甲苯。
5.称量 0.1 毫摩尔的硝酸银,并将其转移到标记为 Ag 前体的剩余玻璃瓶中。然后,依次加入 1 ml 油胺和 1 mL 甲苯。
6.用封口膜密封两个小瓶的盖子,并将它们放在具有高功率模式的超声波仪中,以完全溶解所有试剂。
注意:为了快速溶解试剂,在此过程中可以将小瓶加热到大约 40 °C 。 此步骤没有时间限制,但应在注射步骤之前准备好。
7.准备两个隔垫,如图 2 所示。 
注意:使用 12G 针打孔,将巴斯德吸管插入橡胶隔垫。在孔中加入一些乙醇可以使插入更容易。插入后乙醇会蒸发。


 
图 2 。为 Ar 起泡准备的隔垫橡胶。
稍后,将针从隔垫(顶部)中取出以插入热电偶。


8.装有反应原料(步骤A2制备)的三颈圆底烧瓶放在套式加热器上。
9.将捕集球连接到烧瓶的中心颈部,并用夹子夹住它们。然后,插入冷凝器管,并将冷凝器的另一端连接到含有液体石蜡的捕集器(图 3)。
注意:回流冷凝器使用空气,没有自来水。




图 3. (A) 用于制备 MPNP 的实验装置的插图和 (B) 照片。


10.使用图 2 所示的隔膜密封剩余的两个开口颈部。
注意:用于 Ar 鼓泡的巴斯德吸管的尖端应浸入反应溶液中,但不要接触磁力搅拌棒。
11.以 150 rpm 的速度打开磁力搅拌器,并通过巴斯德吸管以 0.35 L/min 的流速泵入 Ar 气体。气体通过 12G 针流出。静置 5 分钟,完成用 Ar 气置换烧瓶中的气氛。
12.取下针头,插入热电偶。
13.打开温度控制器,并将温度设置为 100 °C。
注意:由于溶液在室温下的高粘度,搅拌速度不能立即设置为 500 rpm。因此,在温度升高的同时,慢慢提高搅拌速度。在 50°C 时,搅拌速度可完全设置为 500 rpm。该步骤的升温速率约为12-13 ° C/min 。
14.从温度达到100°C 的那一刻起,保持 10 分钟。在此步骤中,形成了银种子。
注意:可能会观察到过热,其中温度升高到 100°C 以上。稍微降低加热套以减少其与烧瓶的接触会降低温度。
15.通过将温度控制器设置为 300°C ,将温度提高到 250° C。注意:此设置的目的是将温度线性升高到250°C (图 4) 。




图 4。 MPNPs制备过程的温度曲线。


16.在温度升高期间,一旦达到 170°C,使用玻璃注射器和 20 G 针注入预先制备的 Fe 和 Co 前体混合物。
注意:针通过包含热电偶的隔膜插入。以快速的速度逐滴注入溶液,持续 1 分钟。通过稍微降低加热罩以减少与烧瓶的接触,温度应保持在 170°C 左右。从 100°C到 170°C的加热速率约为 12 ° C/min 。
17.一旦温度达到 250°C ,使用玻璃注射器和另一个 20G 针注入预先制备的 Ag 前体。然后,立即将控制器的温度重置为 230°C。保持反应10分钟。
注意:这是最重要的一步。 Ag 前驱体的注入通常在大约 20 秒内完成。它不应小于 10 秒,或长于 30 秒。注射后,稍微降低加热套以减少与烧瓶的接触,以免过热。我们确认反应时间可以延长到 15 分钟,但 MPNP 的质量没有受到影响。从 170°C到 250°C的加热速率约为 9 °C/min 。
18.将温度控制器设置为 0 °C 以停止加热,然后取下地幔加热器。等待反应体系自然冷却,同时继续搅拌和Ar鼓泡。此时,部分合成颗粒会附着在磁力搅拌棒上,但在冷却过程中会再次重新分散。
19.一旦反应溶液的温度低于 70°C ,停止 Ar 气流,并关闭磁力搅拌器。
20.小心拆卸装置,用移液器将反应溶液从三颈瓶中均匀转移到两个 50 mL 离心管中。
21.加入丙酮,将管子填充至 45 mL。然后,使用 Kubota 5910 在 4,640 × g和RT下进行离心5 分钟。
22.丢弃所有上清液,并在每管中加入 400 μL 的己烷,以重新分散 MPNP。
23.使用微移液器将 200 μL 的再分散 MPNP 溶液转移到另外两个 50 mL 管中。随后,总共填充高达 45 mL 的丙酮,并使用 Kubota 5910 在 4,640 × g和RT下进行离心5 分钟。
24.丢弃上清液,将获得的 MPNPs 重新分散在 3 mL 的氯仿中。使用紫外-可见吸收分光光度计通过吸收光谱确定MPNP 分散体的浓度。
注:MPNPs 的浓度使用y = 0.024x的校准曲线确定,其中 y 是 MPNPs 的局部表面等离子共振的吸收峰值, x是 MPNPs 的浓度 (µg/mL)。
25.将获得的 MPNPs 储存在 4°C 的氯仿中,放入带封闭顶盖的玻璃小瓶中。用封口膜密封。


B.聚乙二醇化磷脂中 MPNP 的封装
1.准备一个 50 mL 的双颈圆底烧瓶(图 5A)。
2.将 3 mL 的 MPNPs 以 1 mg/mL 的浓度分散在氯仿中,倒入烧瓶中。随后,在氯仿中加入 1,350 μL 的 18:1 戊二酰 PE(5.5 mM),在氯仿中加入 900 μL 的 PEG350-DOPE(5.5 mM)到分散体中。
3.使用隔膜橡胶密封,将装有巴斯德移液器的隔膜放在中心颈部,将含有针头的隔膜放在倾斜的颈部。
注意:巴斯德移液器的尖端不应浸入分散液中。
4.以 0.5 L/min 的速率泵入 Ar 气体,以完全蒸发氯仿(图 5B)。
注意:在此步骤中,惰性气体可以是 Ar 或 N 2 。




图 5. (A) 玻璃器皿和 (B) 在聚乙二醇化磷脂中封装 MPNP 的实验装置。


5.添加 1.5 mL 的去离子水以重新分散获得的沉淀。将分散液收集到两个 1.5 mL 离心管中。
6.Hitachi CF15RXII高速微型离心机以 1,400 × g和RT离心5 分钟,以消除大的聚集颗粒。
7.小心地将离心管中的上清液转移到两个新的 1.5 mL 管中。然后,使用CS100FNX超速离心机在 86,600 × g和4°C下离心10 分钟,以去除空胶束。
注意:在此步骤中,如果 MPNP 未完全收集,则将离心速度提高到 100,000 × g。
8.丢弃上清液,将获得的颗粒重新分散在 1 mL 的去离子水中。
9.从吸收光谱中确定磷脂包裹的 MPNPs 的浓度。


C.使用 EDC 偶联反应偶联 aDxt
1.将 20 mL 的 MES 缓冲液(pH ~6)添加到包含磁力搅拌器的 100 mL 单颈圆底烧瓶中。
2.将磁力搅拌器设置为 600 rpm。
3.在烧瓶中加入 1 mL 的磷脂封装 MPNPs 分散体(1 mg/mL)。然后,在去离子水中依次添加 125 μL 的 EDC(200 mM)和 MES 缓冲液中的 250 μL NHS(200 mM)。将其留在RT 30 分钟。
注意:从冰箱中取出后,使用前将 EDC 和 NHS 平衡至 RT。
4.将得到的反应混合物转移到 20 × 1.5 毫升管。
5.CS100FNX在 86,600 × g和4°C 下离心10 分钟,小心去除上清液。
6.使用微移液器收集并将获得的颗粒重新分散在 1 mL 的 PBS 中。
7.准备一个 100 mL 的单颈圆底烧瓶,其中含有 19 mL 的 PBS。
8.将 50 mg aDxt 加入烧瓶中的 PBS 溶液中(步骤 C7 中制备),并在磁力搅拌下等待其完全溶解。
9.将 MPNPs 的 PBS 分散体(在步骤 C6 中制备)添加到 aDxt 的 PBS 溶液中(在步骤 C8 中制备)。然后,在 RT 下保持反应 90 分钟。
10.将获得的混合物转移到 20 × 1.5-mL 管中。然后,使用CS100FNX在 60,000 × g和4°C 下离心10 分钟。在 1 mL 的 PBS 中收集并重新分散获得的颗粒。
11.确定分散体中 aDxt 共轭 MPNP (aDxt-MPNP) 的浓度,使用 吸收光谱。


D.用于研究 MPNPs 细胞内运输的脉冲追踪实验
笔记:
a.在脉冲追踪实验中,脉冲是 aDxt-MPNPs 与 COS-1 细胞孵育一段时间(t load )。在加载步骤之后,去除未掺入细胞中的过量 aDxt-MPNP。细胞在新鲜培养基中进一步培养一段给定的时间,t追逐。本实验的目的是找到最佳的t追逐,以使 aDxt-MPNPs 通过内溶酶体途径到达溶酶体区室。
b.这些实验是在无菌条件下的洁净工作台上进行的。
1.将 10 – 20 个灭菌的圆形盖玻片放入 10 厘米的培养皿中。
2.在 10 厘米的盘子中加入 5 mL 的 0.01% PLL 溶液,并使用镊子在 RT 的 PLL 溶液中浸入圆形盖玻片 5 分钟。
3.取出 PLL 溶液,用铝箔覆盖,铝箔部分打开,在干净的工作台上自然干燥过夜。
4.第二天,使用 5 mL 的 PBS 缓冲液清洗 PLL 涂层盖玻璃三次。
5.使用镊子将四个盖玻璃放在 24 孔板的每个孔中,这对应于1 小时、2 小时、4 小时和 7 小时的不同t追逐值。
注:盖玻片的数量根据被调查的数量增加。在 COS-1 细胞的情况下,最大 t追踪在 7 小时进行。但需要注意的是,t追逐的长度取决于不同的细胞系。因此,本实验的孵育时间可以轻松定制。
6.°C下 5% CO 2下在 DMEM (+10% FBS) 中孵育过夜。
7.提前在明场显微镜(Keyence,型号: BZ-X810)下检查细胞的健康和融合情况。
8.第二天,取出培养基,在 RT 处用 500 μL 的 PBS 清洗细胞。
9.°C下在 5% CO 2下孵育 30 分钟。
10.在完成饥饿过程前大约 10 分钟,将 MPNPs 添加到 DMEM(+10% FBS)中,以制备浓度为 100 μg/mL 的 MPNPs 分散体。
11.饥饿后立即用 500 μL 的 MPNPs 分散在 DMEM(+10% FBS)(在步骤 D10 中准备)替换没有 FBS 的 DMEM,并在 37 °C下在 5% CO 2下孵育t负载= 1 小时。
12.孵育 1 小时后,去除分散液,并用 500 μL 的 PBS 洗涤一次。然后,添加 500 μL 的预热 DMEM(+10% FBS),并在 5% CO 2下在 37 °C下孵育t追逐:0 小时、2 小时、4 小时和 7 小时。
注意:t追逐在不同的细胞系中会有所不同。因此,可以适当地定制潜伏期。
13.完成t追逐后,用 500 μL 的 PBS 洗涤 3 次,并在 RT 下向每个孔中加入 500 μL 的 4% PFA 15 分钟。
注意:由于 PFA 是一种有毒化学物质,工作人员必须穿实验室外套和化学防护手套。此步骤应在配备通风系统和防护窗扇的洁净工作台中进行。此外, 使PFA溶液远离火焰或热源。 PFA 应作为危险废物妥善处置。固定后,可在洁净工作台外进行以下步骤。不需要无菌条件。
14.用 500 μL 的 PBS 洗涤 3 次,加入 500 μL 的 50 μg/mL 地黄皂苷-PBS 对每个孔进行渗透,然后等待 5 分钟。
15.用 500 μL 的 PBS 洗涤 3 次,在每口井中加入 500 μL 的 50 mM NH 4 Cl-PBS,等待 10 分钟。
16.用 500 μL 的 PBS 洗涤 3 次,每孔加入 500 μL 的 3 wt% BSA-PBS,等待 30 分钟进行封闭。
17.对于每口井,加入 500 μL 的 3 wt% BSA-PBS,其中含有 2 μL 的 AF647@CD107A 用于染色溶酶体,并添加 0.25 μL 的 100 μg/mL DAPI 用于染色细胞核。在室温下等待 1 小时,或在 4°C 下过夜。
18.用 500 μL 的 PBS 洗涤三次。对于每个洗涤步骤,加入 PBS 后等待 5 分钟。
19.将一滴防褪色封固剂加到白色载玻片磨边上,用镊子小心地取下盖玻片,然后将其放在玻璃基板上进行观察,使细胞表面与封固剂接触。确保没有气泡并在必要时去除多余的液体。
20.将它放在黑暗的地方几个小时,直到它完全干燥。然后,使用 CLSM观察样本。


E.在 CLSM 下观察加载 MPNPs 的细胞
1.选择 405、473 和 635 nm 激光器分别用于激发 DAPI、aDxt-MPNP 和 AF647。
2.对于 DAPI 染料,选择屏障滤光片 (BA) 435 – 455 nm。
3.对于 aDxt-MPNPs 的等离子体散射信号,请选择无屏障滤波器,因为与荧光染料不同,来自 aDxt-MPNPs 的散射信号具有与激光波长相同的波长。
4.对于 AF647 染料,选择 BA 655–755 nm。
5.捕获超过五个不同随机选择区域的 CLSM 图像。将 DAPI 信号分别记录到等离子体散射信号和 AF647 荧光(图 6)。
6.使用 ImageJ 软件确定阈值 Manders 的共定位系数 ( R t ),对 aDxt-MPNP 和溶酶体进行共定位分析。




图 6。 由 CLSM 捕获的 0 h、2 h、4 h 和 7 h 的不同t追踪值下加载 aDxt-MPNP 的 COS-1 细胞的 CLSM 图像(比例尺:20 µm)。
如文中所述,细胞核(蓝色)和溶酶体(红色)分别被 DAPI 和 AF647 染色。通过等离子体散射信号观察到 aDxt-MPNP。使用 ImageJ 获得合并的图像。经Le等人的许可改编。 ( 2022 年) 。版权所有 2022 美国化学学会。


F.aDxt-MPNPs 在溶酶体中的积累、均质化和溶酶体的磁分离
1.种子 2 × 10 6 COS-1 细胞/培养皿用于两个 10 厘米培养皿,并在 DMEM(+10% FBS)中孵育 24 小时。
2.提前在明场显微镜(Keyence,型号: BZ-X810)下检查细胞的健康和融合情况。
注意:通过使用明场显微镜确认它们在细胞培养皿上的贴壁状态来检查 COS-1 细胞的健康状况。此外,还同时检查了污染的可能性。在这个特定的实验中,细胞的汇合率估计低于 80%。但是,它会根据细胞类型而改变。所需的细胞汇合率为 70% 至 80%。如果细胞很好地粘附在培养皿上而没有被污染,并且融合度在 70-80% 左右,则可以进行下一步。
3.对于细胞饥饿,在每个细胞培养皿中加入 5 mL 不含 FBS 的预热 DMEM,并在 37°C、5% CO 2下孵育30 分钟。
4.在完成饥饿过程前约 10 分钟,将 MPNPs 添加到 DMEM(+10% FBS)中,以制备 10 mL 的 MPNPs 分散体,浓度为 100 μg/mL。
5.DMEM (+10% FBS)中的 MPNPs(在步骤 F4 中制备)替换没有 FBS 的DMEM ,并在 37ºC 下在 5% CO 2下孵育8 小时。
注:在本研究中,我们选择 t load = 8 小时装载。 t load强烈影响溶酶体的分离产量。根据 MPNP 对细胞的细胞毒性,该参数可以延长。
6.丢弃含有培养基的 aDxt-MPNPs,并用预热的 PBS 洗涤一次。
7.添加 5 mL 的 DMEM(+10% FBS),并进一步孵育t追逐= 7 小时。最佳t追逐已经在脉冲追逐实验部分确定。根据细胞类型,此参数可能会有所不同。
8.将必要的设备,包括磁柱、MidiMACS 分离器、带 23G 针头的 2.5-mL 注射器(带针头的 Terumo 注射器 2.5mL 23G 蓝色)和 5-mL 试管放入温度保持在 4° 的冷藏室中C,在脉冲追踪实验前至少 30 分钟,以平衡温度。如果没有冷藏室,请使用冰盒来存放设备。
9.丢弃培养基,用 PBS 清洗细胞。
10.在每个培养皿中加入 1.5 mL 的冷 PBS,并将它们放在冰上。
11.使用 Cell Lifter 刮掉细胞,并将它们从两个培养皿转移到 15 mL 离心管中。使用带有 ST-720 摆动斗式转子的 Kubota 5910在 190 × g和 4°C 下离心 4 分钟。
注意:在此步骤中,可以通过细胞颗粒的颜色对颗粒吸收量进行定性评估(图 7)。颜色越深,内化的粒子数量越多。如果细胞颜色仍然是白色,则意味着吸收效率非常低。溶酶体的分离可能会失败。
12.弃去上清液,加入 1 mL 冰冷的 PIS 以重新悬浮细胞颗粒。然后,转移到 5 mL 管中,并保存在冰箱中。
注:此步骤后,实验在低温室继续进行,温度保持在 4°C。




图 7。 aDxt-MPNPs 加载 COS-1 细胞的照片, t负载= 8 小时, t追逐= 7 小时。


13.使用带有 23G 针头的 2.5 mL 注射器,反复(15 次)将细胞悬液通过注射器,以使细胞均质化。
注意:必须通过实验确定最佳通道数(图 8)。均质效率低会明显影响溶酶体分离的产量。相反,通过增加通道数来提高均质化效率也可能导致溶酶体被破坏。因此,在这项研究中,留下了一小部分未破碎的细胞或大细胞碎片。




图 8。 相衬模式下COS-1细胞的明场图像。
(A) 之前和 (B) 使用带有 23G 针头 (15通道) 的注射器进行均质化之后。在均质化之前,可以清楚地看到细胞被明亮的光晕包围的高密度暗区。均质化后,细胞数量减少,由于细胞膜破裂,细胞混合物变成浆液。因此,明亮光晕的数量显着减少。仍然可以观察到一小部分未破碎的细胞或浆液中的大细胞碎片。比例尺,100 µm。 


14.在 MidiMACS 分隔符中放置一个 MS 列。
注意:本实验也可以使用另一种 MACS ®色谱柱,例如 LS 色谱柱。
15.通过添加 1 mL 的 PIS 来平衡 MS 色谱柱。丢弃流通。
16.将细胞裂解物(在步骤F13中制备)转移到 MS 色谱柱,并让细胞裂解物通过色谱柱。磁性部分将被困在色谱柱内,而非磁性部分将通过色谱柱。
17.丢弃流过。使用 1 mL 的 PIS 清洗色谱柱两次,以进一步消除未结合的材料。
18.从 MidiMACS 分离器中移除 MS 色谱柱。
19.添加 0.5 mL 的 PIS,并插入柱塞以在 1.5 mL 微管中收集含有溶酶体的磁性部分。再次重复此步骤。
20.将获得的悬浮液以 5,000 × g离心 和 4°C 10 分钟,使用 MDX-310系统。
注意:此步骤是去除分离部分中剩余的可溶性蛋白质。
21.丢弃上清液,将获得的溶酶体颗粒重新分散在 100 μL 的 PIS 中。
注意:如果对分离的溶酶体部分进行蛋白质组分析,则将颗粒重新悬浮在 50 mM 三乙基碳酸氢铵中。


数据分析


aDxt-MPNPs 和溶酶体的共定位分析使用 ImageJ 进行。首先,打开图像,然后选择“插件” “分割” “颜色聚类”。之后,在新窗口的“通道”部分中,为图像选择合适的颜色。对于这种特定情况,aDxt-MPNP 和溶酶体的图示颜色分别为绿色和红色。接下来,按“运行”,然后选择“显示结果”,得到分割后的图像。请注意,一次只能处理一张 aDxt-MPNP 或溶酶体的图像(图 9)。获得分割图像后, R t 表示与 aDxt-MPNP 重叠的溶酶体的百分比。要打开 aDxt-MPNP 和溶酶体的彩色聚类图像,请选择“分析” “共定位” “Coloc 2”。在新窗口中,选择通道 1 的 aDxt-MPNPs 图像和通道 2 的溶酶体图像,然后选中 Manders 相关框, 并按 OK(图 10)。对来自每个实验条件的五对图像重复此步骤。






图 9。 使用颜色聚类对 CLSM 图像进行分割。
(A) 选择“插件”>“分割”>“颜色聚类”。 (B) 对于 aDxt-MPNP 图像,纳米粒子的图示颜色为绿色,因此,选择绿色(在通道部分),然后单击“运行”。对染色溶酶体的 CLSM 图像重复此步骤,并相应地选择适当的颜色进行分割。




在 ImageJ 软件中使用 Coloc 2 插件确定R t的过程。
(A) 在前面的步骤中打开两个分割图像,选择“分析”>“共定位”>“Coloc 2”。 (B) 在新窗口中,选择通道 1 和通道 2 的图像。然后,检查 Manders 的相关性框,然后单击确定。


完成图像分析后,可以构建R t与孵育时间的关系图(图 11A)。孵育时间是t load和t chaos之和。随着 aDxt-MPNPs 被转运到溶酶体, R t值增加。然而,由于 CLSM 图像的空间分辨率有限, R t值是饱和的。从该图中,可以确定值tchas 。通常情况下,追 在R t达到一个平台期后选择。这背后的原因是,在内溶酶体途径中,晚期内体与溶酶体融合,这也可能导致 aDxt-MPNPs 与溶酶体的高度共定位。然而,建议在获得分离的溶酶体部分后,应使用西方印迹分析评估晚期内体的水平(图 11B)。如果晚期内体仍然存在,则需要进一步延长t追踪。






图 11。 aDxt-MPNPs 与溶酶体的延时共定位以及细胞裂解物、PS 和 NS 部分的蛋白质印迹。 
(A) R t与孵化时间的关系图。 ADxt-MPNPs 在溶酶体中的积累表现为R t随着时间的增加。 (B) 分离溶酶体部分的蛋白质印迹。 PS:正选择(磁性分数); NS:负选择(非磁性部分); GAPDH:甘油醛-3-磷酸脱氢酶(胞质蛋白作为对照); LAMP2:溶酶体相关膜蛋白 2(溶酶体标记),Rab7:晚期内体标记蛋白。经Le等人的许可改编。 ( 2022 年) 。版权所有 2022 美国化学学会。


食谱


1.MES 缓冲液(0.1 M,pH ~6)
将 3.90 g MES 溶解在 180 mL 的去离子水中。使用 pH 计监测溶液的 pH 值,然后使用微量移液器吸取 10 N 氢氧化钠水溶液,将溶液的 pH 值调节到大约 6。然后,加水至 200 mL。使用前通过 0.2- µ m 过滤器过滤对溶液进行消毒。存放在深色瓶子中。
2.PBS缓冲液
将 9.6 克 PBS 溶解在 1 L 的去离子水中。该溶液在使用前应通过高压灭菌器进行灭菌。将溶液储存在 4°C。
3.洋地黄皂苷溶液
在 500 μL 二甲基亚砜 (DMSO) 中溶解 25 mg 地黄皂苷。将溶液分成微管,15 μL/管。将溶液储存在 -20°C 。用 PBS 稀释,用于 透化。
4.氯化铵溶液
在 60 mL 的 PBS 缓冲液中溶解 0.160 g NH 4 Cl。将溶液储存在 4°C 。
5.DAPI染色液
从市售产品中制备浓度为 100 µg/mL 的 DAPI 原液,储存在 4°C 的冰箱中。对于细胞核染色,将原液进一步稀释 2000 倍。
6.蛋白酶抑制剂溶液 (PIS)
a.在 50 mL 管中准备 20 mL 的 PBS。
b.中加入 20 μL的 0.1 M苯甲基磺酰氟和 100 μL的蛋白酶抑制剂混合物。苯甲基磺酰氟和蛋白酶抑制剂混合物的稀释倍数分别约为 1000倍和 200倍。
c.使用涡流均匀地分散溶液。然后,将溶液保存在冰盒中。
注意:此溶液应在使用时准备好。不建议长期存放。抑制剂混合物的组成为: 0.1 mg/mL 亮肽素半硫酸盐一水合物; 0.14 毫克/毫升胃酶抑素A; 14 mg/mL N-对甲苯磺酰基-L-苯丙氨酸氯甲基酮; 15 mg/mL Nα-对甲苯磺酰基-L-精氨酸甲酯盐酸盐; 0.4 毫克/毫升抑肽酶;溶解在 DMSO 中的 32 mg/mL 苯甲脒。抑制剂混合物可以提前制备并储存在 -20°C 的小管中。


致谢


该协议源自原始研究论文 Le等人。 “完整溶酶体的快速温和分离 使用磁-等离子体混合 纳米粒子” ACS Nano 2022 年 1 月 3 日; 16(1):885-896。 doi:10.1021/acsnano.1c08474(Le等人,2022)。这项工作的部分资金来自日本社会的青年科学家资助 科学促进会(授权号 21K14506)给 MT


利益争夺


作者声明没有竞争利益。


伦理


本研究未使用人类或脊椎动物受试者。


参考


1.De Duve, C.、Pressman, BC、Gianetto, R.、Wattiaux, R. 和 Appelmans, F. (1955)。组织分级研究。 6. 大鼠肝组织中酶的细胞内分布模式。 生化杂志 60(4):604-617。
2.Le, TS, Takahashi, M., Isozumi, N., Miyazato, A., Hiratsuka, Y., Matsumura, K., Taguchi, T. 和 Maenosono, S. (2022)。使用磁性-等离子体混合纳米粒子快速温和地分离完整的溶酶体。 ACS 纳米16(1):885-896。
3.Milosevic, AM, Rodriguez-Lorenzo, L., Balog, S., Monnier, CA, Petri-Fink, A. 和 Rothen-Rutishauser, B. (2017)。通过使用补充方法评估溶酶体中荧光编码纳米颗粒的稳定性。 Angew Chem Int Ed Engl 56(43):13382-13386。
4.Mukherjee, AB、Appu, AP、Sadhukhan, T.、Casey, S.、Mondal, A.、Zhang, Z. 和 Bagh, MB (2019)。溶酶体和神经元蜡样脂褐质糖的新作用。 摩尔神经变性剂14(1):4。
5.Singh, J.、Kaade, E.、Muntel, J.、Bruderer, R.、Reiter, L.、Thelen, M. 和 Winter, D.(2020 年)。通过数据独立采集富集溶酶体的策略的系统比较。 J 蛋白质组研究19(1):371-381。
6.Snipstad, S., Hak, S., Baghirov, H., Sulheim, E., Morch, Y., Lelu, S., von Haartman, E., Back, M., Nilsson, KPR, Klymchenko, AS, et人_ (2017)。标记纳米颗粒:染料泄漏和改变的细胞摄取。 细胞术 A 91(8):760-766。
7.Takahashi, M.、Higashimine, K.、Mohan, P.、Mott, DM 和 Maenosono, S.(2015 年)。磁性-等离子体Ag@FeCo@Ag核-壳-壳纳米粒子的形成机制:事实比虚构更有趣。 CrystEngComm 17(36):6923-6929。
8.Thomsen, T., Ayoub, AB, Psaltis, D. 和 Klok, HA (2021)。聚合物纳米颗粒装饰的 T 细胞的基于荧光和无荧光标记的表征。 生物大分子22(1):190-200。




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引用:Le, T. S., Takahashi, M. and Maenosono, S. (2022). A Robust Nanoparticle-based Magnetic Separation Method for Intact Lysosomes. Bio-protocol 12(13): e4453. DOI: 10.21769/BioProtoc.4453.
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