Macrofungal-Derived Extracellular Vesicle Isolation and Purification Kit

Reagents, Consumables and Equipment Required but Not Provided

1. Instruments and Tools

Fruit knife, juicer, and other tools for macrofungal sample pretreatment and juicing/grinding; electronic balance; water bath; vortex mixer, etc.

2. Consumables

Beakers; centrifuge tubes (50 mL and 1.5 mL); pH test strips or pH meter, etc.

3. Reagents

Ethanol (75%), sterile PBS (1×, MCE Cat. No.: HY-K3005), etc.

Macrofungal Vesicle Extraction

1. Juicer Disinfection

Disassemble the components of the juicer or mortar, spray with 75% ethanol and wipe thoroughly. Place the components in a biosafety cabinet and disinfect by UV irradiation for > 20 min. Reassemble the equipment after disinfection.

2. Sample Pretreatment

Prepare the macrofungal samples according to the kit size and corresponding sample-processing volume.

3. Sample Disinfection

Thoroughly wash the fresh macrofungal sample to remove surface impurities and dry it with a lint-free wipe. Place the sample in a beaker and disinfect by UV irradiation in a biosafety cabinet for > 20 min. Then cut the fungal fruiting body into thin slices suitable for juicing.

Note: Macrofungi generally have relatively low water content, and direct juicing may yield only a limited amount of juice. It is recommended to cut the fruiting body thoroughly into small pieces or thin slices to improve juicing efficiency.

4. Sample Juicing

Transfer the prepared fungal fruiting-body slices to a juicer. It is recommended to juice the sample in several small batches and collect the fungal residue discharged from the outlet.

Note: a. If endotoxin control is required, perform the juicing/grinding procedure in a biosafety cabinet.

b. If the collected fungal residue still contains incompletely disrupted fruiting-body tissue, juice or grind the residue again to facilitate complete release of vesicles from the tissue.

5. Fungal Residue Dispersion

Add PBS (1×, MCE Cat. No.: HY-K3005) to the collected fungal residue while stirring continuously until the residue is fully dispersed in PBS and the resulting mixture has good fluidity.

Note: The amount of PBS should be adjusted according to the viscosity of the tissue components in the fungal residue. It is recommended to add PBS gradually in several small portions while mixing thoroughly and monitoring the dispersion and fluidity of the mixture.

6. Sample Pretreatment

Thaw Extraction Buffer A at 4°C in advance and mix thoroughly. Add an appropriate volume of Extraction Buffer A to the PBS-dispersed fungal residue mixture as shown below.

Note: a. After the first thaw, it is recommended to aliquot Extraction Buffer A appropriately to minimize repeated freeze-thaw cycles.

b. Yellow-brown precipitates may appear after prolonged storage of Extraction Buffer A. This is normal. Mix thoroughly before use to ensure uniform distribution of all components.

7. Mixing and Incubation

Seal the centrifuge tube with sealing film and gently invert 8-10 times to thoroughly mix the sample with Extraction Buffer A. Incubate the mixture in a 37°C water bath for 16 h.

8. pH Adjustment

After incubation, adjust the mixture to pH 7.0 in a biosafety cabinet using citric acid (1%) and NaOH (1 M).

9. Centrifugation for Impurity Removal

1) Low-speed centrifugation: Centrifuge the collected liquid at 5,000 g for 10 min at 4°C and collect the supernatant.

2) High-speed centrifugation: Centrifuge the supernatant at 10,000 g for 20 min at 4°C and collect the supernatant.

10. Filtration

Sequentially filter the collected supernatant through 1 μm and 0.45 μm membrane filters.

Note: If macrofungal vesicles with a particle size of approximately 30-1,000 nm are required, filtration through a 1 μm membrane alone may be performed.

11. Vesicle Extraction

Add an appropriate volume of Extraction Buffer B to the filtered supernatant as shown below.

12. Mixing and Incubation

Seal the centrifuge tube with sealing film and vortex for 1 min to thoroughly mix the sample with Extraction Buffer B. Then incubate the mixture statically at 4°C for 4 h.

13. Vesicle Precipitation

After incubation, centrifuge the mixture at 10,000 g for 1 h at 4°C and discard the supernatant. Centrifuge again at 10,000 g for 2 min at 4°C and discard the supernatant.

Note: The resulting pellet is enriched in macrofungal vesicles. Remove the supernatant as completely as possible to minimize residual liquid.

15. Vesicle Resuspension

Add 400 μL of PBS (1×) to resuspend the pellet. Gently pipette until the pellet is fully dispersed and transfer the suspension to a new 1.5 mL EP tube.

Note: For every 20 mL of macrofungal juice processed, 400 μL of PBS (1×) is recommended for resuspension.

16. Vesicle Collection

Centrifuge the resuspended sample at 12,000 g for 2 min at 4°C and transfer the supernatant to a new centrifuge tube. The resulting supernatant is enriched in macrofungal vesicles.

Note: If a substantial pellet remains after centrifugation, repeat the centrifugation step until no obvious pellet is observed. Retain the supernatant after each centrifugation.

Macrofungal Vesicle Purification and Storage

1. Vesicle Purification

Transfer the collected crude macrofungal vesicle preparation to the upper chamber of the vesicle purification column and centrifuge at 3,000 g for 10 min at 4°C. Collect the liquid at the bottom of the purification column as the purified macrofungal vesicle preparation.

Note: If larger macrofungal vesicles need to be retained, this purification step may be omitted.

2. Vesicle Storage

Aliquot the purified macrofungal vesicles and store at -80°C until use. Avoid repeated freeze-thaw cycles to preserve vesicle integrity and associated biological activity.