Skip to content

In vitro Culture of Dirofilaria immitis L3 - L4 Molt


  • IMDM (Sigma I3390)
  • NCTC-135 Powder (Sigma N3262-10X1L) or NCTC-135 L-glutamine/ HEPES (Sigma N3262)
  • NaHCO3
  • Millipore Water
  • Fetal Bovine Serum (Heat Inactivated)
  • Fungin 1000X Stock (10 mg/mL, Invitrogen ANT-FN-1)
  • Pen/Strep 100X Stock (10,000 U/mL, Sigma P4333-100ML)
  • Gentamicin 100X Stock (10 mg/mL, Sigma G1272-100ML)
  • Ciprofloxacin 1000X Stock (10 mg/mL, Sigma 17850)
  • Optional: Antibiotic-Antimycotic (100X, Gibco 15240096)
  • Optional: HEPES (1M) if using NCTC-135 powder
  • 0.22 µm filter (500 mL, example: VWR 10040-436)


Preparation of culture media

  1. In an autoclaved glass bottle, prepare the following:

    Preparing NCTC if using powder form (⅛th the packet). Store at 4°C for up to one month.

    NCTC Media Component Volume/Quantity (125 mL)
    NCTC-135 Powder 1.16 g
    NaHCO3 0.28 g
    H2O 125 mL
    HEPES (1M) - optional 3.125 mL

    Preparing Molting Media (NCTC-IMDM)

    NI Media Component Volume/Quantity (100 mL)
    NCTC 40 mL
    IMDM 40 mL
    Pen-Strep (100X) 1 mL (10 U/mL final)
    Gentamicin (100X) 1 mL (100 ug/mL final)
    Fungin (1000X) 100 µL (10 ug/mL final)
    Ciprofloxacin (1000X) - optional 100 µL (10 ug/mL final)
    FBS 20 mL (20% final)

    Alternatively, replace P/S and Fungin with 100X Antibiotic-Antimycotic.

  2. Replace cap on media bottle and shake to mix. Filter the media using a 0.22 µm filter.

  3. Label filtered media with: name of media, volume, date prepared, and initials. Store media at 4°C. Media is good for 1 month post-preparation.

Extraction of D. immitis L3 from mosquitoes

Follow this Protocol

Preparing parasites and culture plates (24-well bulk culture)

  1. Wash infective larvae:

    • Transfer infective larvae in minimal amount of RPMI-1640 to a 1.5 mL microcentrifuge tube.
    • Spin the microcentrifuge tube at 1000 x g for 10 minutes.
    • Pipette 100 μL from bottom of tube and transfer to new tube containing 500 μL RPMI.
    • To prevent worm loss, check wash from original tube by pipetting remaining solution into petri dish and examining under dissecting microscope. If a significant number of worms were left behind, pipette into the new tube with NI and spin again.
    • Repeat spin/washing infective larvae in media three times following the steps above.
  2. Under BSC, pipette 200 μL from bottom of microcentrifuge tube and transfer to new petri dish containing pre-warmed, sterile culture media

  3. Prepare 24-well cell culture plates with 800 µL of culture media under sterile conditions. Optional: Add a transwell to each well using a sterile forceps (Fisher Scientific 07-200-154).

  4. Transfer desired number of infective larvae (typically 25) in a volume of 200 µL from petri dish to each well in 24-well plates (total volume = 1 mL). Total of 600 larvae is typically used to fill an entire plate. Larvae are incubated at 37°C with 5% CO2.

  5. Change media under BSC every two days by transferring worms (in transwells) to new wells containing fresh culture media using sterile forceps.

  6. L3 molting should be monitored daily using a compound scope at 40x-200x total magnification.

Note: A majority of D. immitis L3 should molt within the first 2-3 days in culture.

Preparing parasites and culture plates (96-well individual culture)

  1. Prepare a 96-well cell culture plate by adding 200 µL of molting culture media per well under sterile conditions.

  2. Wash freshly extracted L3, as described above.

  3. Transfer one L3 in a volume of 1 µL from petri dish containing culture media and L3 to each well of a 96-well plate.

  4. Place plate containing individually cultured L3 at 37°C with 5% CO2

  5. L3 molting should be monitored daily using a compound scope at 40x-200x total magnification.