Isolation of T. vaginalis EVs

Isolation of Extracellular Vesicles from Trichomonas vaginalis

Abstract

This protocol describes the isolation of extracellular vesicles (EVs) from Trichomonas vaginalis using differential ultracentrifugation. Parasites are first cultured in TYM medium supplemented with heat‑inactivated horse serum until reaching the logarithmic growth phase. To induce EV release under serum‑free conditions, cells are washed and incubated for 4 hours in serum‑free TYM base. The conditioned medium is then clarified by low‑speed centrifugation followed by 1 µm filtration to retain a mixed EV population. EVs are finally pelleted by a single ultracentrifugation step at 100,000 × g. This procedure yields EV preparations suitable for downstream applications such as TEM, proteomic analysis, or functional assays.

Materials and Reagents

Reagents

• Trichomonas vaginalis laboratory strains
• TYM medium prepared according to Diamond (1957)
• Heat‑inactivated horse serum (56 °C for 30 min)
• Serum‑free TYM base (TYM without serum)
• Sterile PBS (for EV resuspension)
• Penicillin/Streptomycin (optional)
• Sterile distilled water
• 70% ethanol (for disinfection)

Equipment

• Beckman Coulter ultracentrifuge
• Type 70Ti rotor (Beckman Coulter)
• Refrigerated tabletop centrifuge (300 × g to 3,000 × g)
• Ultracentrifuge tubes compatible with Type 70Ti
• 15 mL and 50 mL sterile conical tubes
• Biological safety cabinet (Class II)
• Inverted microscope
• Pipettes and sterile tips
• Refrigerated microcentrifuge
• Vortex

Optional:

• Transmission electron microscope (TEM)
• Nanoparticle tracking analysis (NTA) instrument
• BCA or equivalent protein quantification assay

Consumables

• Sterile 1 µm filters
• Serological pipettes
• Sterile culture flasks or screw‑cap tubes
• Parafilm
• Ice buckets

Procedure

A. Culturing T. vaginalis

1. Inoculate T. vaginalis into TYM medium supplemented with heat‑inactivated horse serum.
2. Incubate at 37 °C in closed tubes or flasks to maintain microaerophilic conditions.
3. Grow cells to logarithmic phase (typically 16–24 h).
4. Confirm motility and morphology by inverted microscopy.

B. Preparation for EV Release in Serum‑Free Conditions

1. Transfer cultures to conical tubes.
2. Centrifuge at 300 × g for 10 min at RT to pellet parasites.
3. Discard supernatant and resuspend pellet in serum‑free TYM base.
4. Wash once more with serum‑free TYM to eliminate serum residues.
5. Resuspend cells in serum‑free TYM base at the desired volume.
6. Incubate 4 hours at 37 °C to allow EV release.

C. Clarification of Conditioned Medium

1. Collect the conditioned medium after 4 h.
2. Centrifuge at 300 × g for 10 min at 4 °C to remove cells.
3. Transfer supernatant to clean tubes.
4. Centrifuge at 3,000 × g for 15 min at 4 °C to remove cell debris.
5. Transfer supernatant without disturbing pelleted debris.
6. Filter the supernatant through a 1 µm sterile filter to remove large structures while retaining mixed EVs.

D. Isolation of EVs by Ultracentrifugation

1. Load filtered supernatant into ultracentrifuge tubes compatible with Type 70Ti rotor.
2. Balance tubes precisely (± 0.01 g).
3. Ultracentrifuge at 100,000 × g for 2 h at 4 °C.
4. Carefully discard supernatant; the EV pellet may be barely visible.
5. Resuspend the pellet gently in 50–200 μL sterile PBS, avoiding vortexing.
6. Keep on ice for immediate use or store appropriately.

E. Storage and Downstream Use

• Store EV preparations at 4 °C for up to 48 h.
• For long‑term storage, freeze at –80 °C (avoid freeze–thaw cycles).
• Suitable for:
  • TEM characterization
  • NTA analysis
  • Protein quantification (BCA)
  • Functional assays
  • Proteomics / RNA analysis

Notes

• Ensure serum is thoroughly removed before serum‑free incubation to avoid contamination.
• EV pellets may be invisible; handle gently to avoid losing material.
• Keep samples cold during all steps to preserve EV integrity.
• Use low‑binding tubes if EV recovery is low.