In vitro Culture of Dirofilaria immitis L3 - L4 Molt
Materials
- 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)
Protocol
Preparation of culture media
-
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.
-
Replace cap on media bottle and shake to mix. Filter the media using a 0.22 µm filter.
-
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)
-
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.
-
Under BSC, pipette 200 μL from bottom of microcentrifuge tube and transfer to new petri dish containing pre-warmed, sterile culture media
-
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).
-
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.
-
Change media under BSC every two days by transferring worms (in transwells) to new wells containing fresh culture media using sterile forceps.
-
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)
-
Prepare a 96-well cell culture plate by adding 200 µL of molting culture media per well under sterile conditions.
-
Wash freshly extracted L3, as described above.
-
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.
-
Place plate containing individually cultured L3 at 37°C with 5% CO2
-
L3 molting should be monitored daily using a compound scope at 40x-200x total magnification.