Succulent Plant-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 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. 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 plant samples according to the kit size and corresponding sample-processing volume.

3. Sample Disinfection

Thoroughly wash the plant 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.

Note: a. For grapes or plant samples with relatively thick skins, wash and disinfect thoroughly, then remove the peel and cut the sample into small pieces or thin slices.

b. For potatoes, Rehmannia roots, turmeric and other plant samples with relatively thin skins, wash and disinfect thoroughly and then cut directly into small pieces.

4. Sample Juicing/Grinding

Transfer the prepared plant sample to a juicer for juicing. For plant samples with a relatively firm texture, perform juicing in several small batches to improve tissue disruption and juice release. Collect the plant juice for subsequent use.

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

b. The juicer may continuously 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.

5. 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.

6. 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.

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

Centrifuge the mixture 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 plant 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.

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.