Filarial Parasite Culture

Carry out all work in sterile BSC environment and use sterile technique.

Materials

  • RPMI-1640 (Sigma R8758)
  • Pen-Strep 10,000U/mL (Thermo 15140122)
  • Gentamicin 0.4 mg/µL (Sigma G1272-100ML)
  • FBS (Thermo 16000044)

Brugia spp. parasites (mf, L3, Adults)

Note: Make culture media and scale volume as needed. Media can be stored at 4°C for 1 month. Parasites will typically arrive in 50 mL conical tubes. Culture adult parasites for at least 24 hours prior to downstream use.

Culture Media Component Volume (for 50 mL total)
RPMI-1640 44 mL
Pen-Strep (100 U/mL each) 500 μL of 100X stock (10,000 U/mL each)
Gentamicin (4 μg/μL) 500 uL of 100X stock (0.4 mg/μL)
FBS (10% v/v) 5 mL

Pre-heat RPMI-1640 for 30 min at 37°C. Thaw FBS, Pen-Strep and Gentamicin at room temperature.

  1. Pellet parasites

  2. To pellet mf, spin in benchtop centrifuge (2,000 x g) for 10 min.

  3. To pellet L3 or adults, let settle for 30 min. at room temperature in BSC.

  4. Remove nearly all of the shipping media.

  5. Transfer remaining volume containing the parasites into a new conical tube. Add fresh culture media to tubes (10 mL of media to 15 mL conical; 30 mL of media to 50 mL conical).

  6. Use 15 mL conical tubes for mf and L3.

  7. Use 50 mL conical tubes for adults. If culturing for more than 48 hrs., place 5 adults per well of a 12-well culture plate containing 6 mL of culture media per well or 3 adults per well of a 12-well culture plate containing 3 mL of culture media per well.

  8. Place labeled tubes (with caps loosened) or cell culture plates in 37°C + 5% CO2 incubator.

  9. Remove 90% of media every 24 hours and replace with fresh media.

D. immitis parasites (L3s, Adults)

Note: Make culture media and scale volume as needed. Estimate ~ 4 mL media / adult. Media can be stored at 4°C for 1 month. pH to 7.2 with NaOH droplets or HCl using pH meter. Vacuum filter (0.22 μm filter).

Culture Media Component Volume (for 50 mL total)
RPMI-1640 44 mL
Pen-Strep (100 U/mL each) 500 μL of 100X stock (10,000 U/mL each)
HEPES (20 mM) 500 μL of 2M HEPES
D-glucose (1% w/v) 0.5 g
NaHCO3 (1% w/v) 0.5 g
FBS (10% v/v) 5 mL

Follow protocol for Brugia, with the following modifications: Culture adults at 39°C in 250 mL cell culture flasks with caps loosened if unfiltered flasks are used. Culture L3s at 39°C in labeled 15 mL conical tubes (with caps loosened).