Filarial parasite culture. Carry out all work in a sterile BSC environment using sterile technique.

Materials

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

Brugia spp. (mf, L3, adults)

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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.

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Adult and mf media

Component	Volume (per 100 mL total)
RPMI-1640	88 mL
Pen-Strep (100 U/mL each)	1 mL of 100X stock (10,000 U/mL each)
FBS (10% v/v)	10 mL

L3 media

Component	Volume (per 100 mL total)
RPMI-1640	77.86 mL
Pen-Strep (100 U/mL each)	1 mL of 100X stock (10,000 U/mL each)
FBS (10% v/v)	10 mL
Gentamicin (40 μg/μL)	400 μL of 10 mg/μL stock
Fungin (10 μg/μL)	100 μL of 1000X stock (10 mg/μL)
Amphotericin B (2.5 μg/mL)	1 mL of 100X stock (250 μg/mL)
Ciprofloxacin (2 μg/mL)	20 μL of 10 mg/mL
Fortaz (2 μg/mL)	20 μL of 10 mg/mL

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Check before use — two problems in this table, carried over from the source.

The listed volumes sum to about 90.4 mL, not the stated 100 mL total. Either a component is missing or the RPMI volume is wrong.

The gentamicin and Fungin stock concentrations are given in mg/μL, which would be impossibly concentrated — these are almost certainly meant to be mg/mL. Confirm against the vendor stocks before making this media.

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Protocol

Pre-heat RPMI-1640 for 30 minutes at 37°C.

Pellet parasites.
- To pellet mf, spin in a benchtop centrifuge at 2,000 × g for 10 minutes.
- To pellet L3s, transfer parasites to a 15 mL tube and let them settle at room temperature.
Remove nearly all of the shipping media.
Transfer the remaining volume containing the parasites into a new conical tube. Add fresh, warm culture media — 10 mL to a 15 mL conical, 30 mL to a 50 mL conical.
- Use 15 mL conical tubes for mf and L3.
- Use 50 mL conical tubes for adults.
Place labeled tubes, with caps loosened, or cell culture plates in a 37°C + 5% CO₂ incubator.
Remove ~90% of media every 24 hours and replace with fresh media.