<aside> 🚨

BSL-2 work. If working with Brugia-infected mosquitoes, all members of the Zamanian and Bartholomay labs must be notified 24 hours in advance. The screened-in room must be reserved and scheduled on the Google Calendar Injection Schedule prior to BSL-2 work. Before bringing the mosquitoes out of the insectary, place BSL-2 signs on all entrances to the lab and to the screened dissection room. Put on PPE: lab coat, goggles, and gloves.

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A. Bulk extraction of L3

Materials

• RPMI-1640 (Sigma-Aldrich R8758)
• 2.5 inch strainer (B00428M7OI)
• Porcelain mortar and pestle
• Ziploc disposable plastic dishes, 8 oz, top cut off
• Pen-Strep 100X stock (10,000 U/mL)
• Ice bucket

Bulk extraction materials

Methods

1. Make 500 mL RPMI + P/S. Place 250 mL in the fridge and 250 mL at 37°C.

2. Cold anesthetize the mosquitoes by placing the cartons in a 4°C refrigerator for about 2–4 minutes, or until knocked down. You can put up to ~600 mosquitoes in the mortar, so combine cartons as needed. When all mosquitoes are knocked down, quickly transfer them from the carton to the mortar.

   <aside> ⏱️

   L3 health after extraction is directly related to how long they are cold inside the mosquitoes. Be careful not to leave infected mosquitoes in the fridge too long — check the carton every 30 seconds. Note that anesthetized mosquitoes can still cling to the side of the cardboard.

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   Mosquitoes transferred to the mortar

3. Using the pestle, gently tap the mosquitoes to disrupt the cuticle. Do not grind or over-tap — more is not better. Gently tap until only a few intact mosquitoes remain.

   

   Tapping with the pestle

4. Transfer the mosquitoes to the mesh strainer and place the strainer into a Ziploc plastic dish containing cold RPMI + P/S, about half full of media. Dip the strainer up and down 3–5 times to remove scales, eggs and other debris — the media should appear cloudy. Do not dip too much, as you can lose the L3s in the strainer.

   <aside> ❓

   Needs clarification. The source here reads "cold RPMI + P/S + G". The "G" component is never defined anywhere in the protocol — possibly gentamicin or glucose. Confirm before use.

   </aside>

   

   Strainer dipped in cold media

5. Discard the media and add clean cold RPMI + P/S to the dish, half full, and dip up and down 3–5 times to further rinse the L3s in the strainer.

6. Transfer the strainer of L3s to a clean second dish containing warm RPMI + P/S — 40–50 mL, enough to cover the L3s — and incubate at 37°C for Brugia spp. or 39°C for Dirofilaria for 30 minutes. This allows the L3s to migrate out of the strainer and into the warm RPMI-1640.

   

   Incubating in warm media

7. Remove the strainer from the dish and transfer all L3s that have migrated into the warm media into a Petri dish containing a small amount (~20 mL) of fresh warm RPMI + P/S.

8. Count the total number of L3s harvested and the number of mosquitoes crushed, then aliquot L3s as necessary. L3s should be transferred to complete L3 media for incubation — see Parasite Culture.

9. Once the extractions are complete, rinse and wash all materials used. Dispose of the used plates in the biohazard bin. Do not throw away the sieve or Ziploc dishes.