Extraction and Isolation of Extracellular Vesicles From Piper betle Leaves Using the Apoplastic Fluid Washing and Size Exclusion Chromatography Method

Materials and reagents

Biological materials

1. Piper betle leaves from the family Piperaceae; young to mature leaves, aged 20-30 days old

Note: Young to mature Piper betle leaves have light to deep green–colored leaves (refer to Figure 1A, B), besides being soft to the touch instead of feathery, and can be bent without breaking. The reason why age matters in choosing the leaves is because of the integrity level of the cell wall. In young to mature leaves, cell walls are less developed, thus better in permitting the infiltration of buffer with gentle pressure.

Figure 1. Harvesting Piper betle leaves. (A) Propagation and cultivation conditions of Piper betle. The plant is propagated vegetatively using stem cuttings containing 2–3 nodes, planted on a raised bed at 60–90 cm spacing with 2 m of supporting pole for climbing, and grown under partial shade of the black net. (B) The Piper betle is supplied with NPK fertilizer to support healthy leaf growth. (C) The plant can reach up to 2 m high after 6 months, when it is ready to be harvested. (D) The farmer is instructed to cut the desired leaves at the petiole with a clean knife.

Reagents

1. Phosphate-buffered saline (PBS), pH 7.4 (Biomedia, catalog number: 820301-1); store at 4 °C

2. 70% ethanol (EtOH) (Chemiz, CAS number: 64-17-5); store at room temperature (RT) in a flammable liquid storage cupboard

Caution: This chemical is flammable.

3. Sepharose^TM CL-2B (Cytiva Sweden AB, catalog number: 10346215); store at RT away from direct sunlight and heat

Caution: This chemical is flammable.

4. Milli-Q (Merck Millipore); keep at 4 °C for the purpose of this protocol; store at RT if not used

Laboratory supplies

1. Glass chamber A (height: 220 mm; diameter: 190 mm; thickness: 5 mm) (PLT Scientific Sdn Bhd)

Note: This glass chamber was ordered and modified based on our needs. You may need a bigger or smaller chamber depending on the size of your leaves, vacuum chamber, vacuum pump, and experiment scale.

2. Glass chamber B (height: 200 mm; diameter: 150 mm; thickness: 5 mm) (PLT Scientific Sdn Bhd)

Note: This glass chamber was ordered and modified based on our needs. You may need a bigger or smaller chamber depending on the size of your leaves, vacuum chamber, vacuum pump, and experiment scale.

3. Parafilm M All-Purpose Laboratory Film (American Can, catalog number: LMA-PM996)

4. 50 mL conical centrifuge tubes (Biomedia, catalog number: 940204)

5. 20 mL needleless syringe (Terumo, catalog number: 250203B)

6. Pipette tips, 200 and 1,000 μL

7. Econo-Pac^® Chromatography Columns kit (Bio-Rad, catalog number: 7321010)

8. Serological pipette, 10 and 25 mL (Bio-Rad, catalog numbers: 031024CM09, 051922CM08)

9. Blunt forceps (LLG Labware, catalog number: 4008476)

10. 500 mL glass beaker (waste container)

11. Cling wrap (Analisa Resources, catalog number: 39201025)

12. Amicon^® Ultra Centrifugal Filter, 10 kDa MWCO (Millipore, catalog number: UFC9010)

13. Eppendorf Safe-Lock^® tubes biobased 1.5 mL (Eppendorf, catalog number: 0030123093)

Equipment

1. Vacuum chamber and pump (HEXO, model: RS-1.5)

Note: The pump oil may not be provided and needs to be bought separately, if not provided by the manufacturer.

2. Refrigerated centrifuge (Centrifuge 5804 R) (Eppendorf, catalog number: 5805FM568504) (fixed-angle centrifuge)

3. Tabletop centrifuge (Trabota, model: J74276-M000) (fixed-angle centrifuge)

4. Micropipette 20–200 μL (Joanlab, model: 01235556)

5. Micropipette 100–1,000 μL (Joanlab, model: 01242536)

6. Analytical laboratory scale (OHAUS, model: PA214)

7. Freezer (-80 °C)

8. Retort stand or box with holes

9. Electronic pipette with stand (Joanlab, model: EP100Pro)

10. Vegetable washing basket (KECH, catalog number: 1079134)

Procedure