Dry Plant-Derived Extracellular Vesicle Isolation and Purification Kit

Reagents, Consumables and Equipment Required but Not Provided

1. Instruments and Tools

Fruit knife, juicer, mortar and other tools for plant 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.

Plant Vesicle Extraction

1. Disinfection of Juicer/Mortar

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 dried plant samples according to the kit size and corresponding sample-processing volume. For large plant samples, cut them into small pieces in advance to facilitate complete soaking and subsequent juicing/grinding.

3. Sample Disinfection

Place the dried plant sample to be extracted in a beaker and disinfect it by UV irradiation in a biosafety cabinet for > 20 min.

4. Sample Soaking

Add an appropriate volume of sterile PBS (1×, MCE Cat. No.: HY-K3005) to the beaker, ensuring that the plant sample is completely immersed. After the sample is fully soaked, remove the soaked plant material and retain the soaking solution for subsequent use.

Note: Soaking time varies considerably among different plant samples. Determine an appropriate soaking time according to plant type, tissue characteristics and sample size. Dried flowers generally require a shorter soaking time, whereas seeds, roots and rhizomes generally require a longer soaking time.

5. Juicing/Grinding

Transfer the soaked plant sample to a juicer or mortar and add an appropriate amount of soaking solution for juicing or grinding until the plant tissue is sufficiently disrupted and the plant juice is largely separated from the plant residue. Collect the plant juice for subsequent use.

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

b. The juicer may generate heat during operation. It is recommended to limit each juicing cycle to within 1 min to minimize the potential effect of temperature increases on plant vesicles.

c. When using a mortar, thoroughly grind the plant tissue first and then add the soaking solution for further grinding.

6. Centrifugation for Impurity Removal

1) Low-speed centrifugation: Centrifuge the collected plant juice 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 10 min at 4°C and collect the supernatant.

7. Supernatant Pretreatment

Thaw Extraction Buffer A at 4°C in advance and mix thoroughly. Add an appropriate volume of Extraction Buffer A to the plant juice after centrifugation, 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.

8. 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.

9. pH Adjustment

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

10. Centrifugation for Impurity Removal

Centrifuge the mixture at 10,000 g for 20 min at 4°C and collect the supernatant.

11. Filtration

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

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

12. Vesicle Extraction

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

13. 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.

14. 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 plant 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 plant 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 plant-derived 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.

Plant Vesicle Purification and Storage

1. Vesicle Purification

Transfer the collected crude plant 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 plant vesicle preparation.

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

2. Vesicle Storage

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