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.
Recipe for adult and mf media
| Culture Media Component | Volume (for 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 |
Recipe for L3 media
| Culture Media Component | Volume (for 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) |
| Ciproflaxin (2 μg/ml) | 20 μL of 10 mg/mL |
| Fortaz (2 μg/mL) | 20 μL of 10 mg/mL |
|Pre-heat RPMI-1640 for 30 min at 37°C.
-
Pellet parasites
-
To pellet mf, spin in benchtop centrifuge (2,000 x g) for 10 min.
-
To pellet L3s, transfer parasites to a 15 mL tube and let settle at room temperature.
-
Remove nearly all of the shipping media.
-
Transfer remaining volume containing the parasites into a new conical tube. Add fresh, warm culture media to tubes (10 mL of media to 15 mL conical; 30 mL of media to 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 37°C + 5% CO2 incubator.
-
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).