In vitro culture of the 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 (1 M) if using NCTC-135 powder
0.22 μm filter (500 mL, e.g. VWR 10040-436)

Preparation of culture media

NCTC from powder (1/8th of the packet). Store at 4°C for up to one month.

Component	Volume/quantity (125 mL)
NCTC-135 powder	1.16 g
NaHCO₃	0.28 g
H₂O	125 mL
HEPES (1 M) — optional	3.125 mL

Molting media (NCTC-IMDM)

Component	Volume/quantity (100 mL)
NCTC	40 mL
IMDM	40 mL
Pen-Strep (100X)	1 mL
Gentamicin (100X)	1 mL (100 μg/mL final)
Fungin (1000X)	100 μL (10 μg/mL final)
Ciprofloxacin (1000X) — optional	100 μL (10 μg/mL final)
FBS	20 mL (20% final)

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