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L3 Emergence Assay

Adapted from the Povolones lab - Original publication in Parasites & Vectors - Example of the assay in action in Veterinary Parasitology - Detailed protocol from


  • warm RPMI-1640 (Sigma R8758)
  • dH2O
  • 70% ethanol
  • 96-well plates used for ImageXpress (USA Scientific 5665-5180Q)
  • Extraction toolbox
  • 2, 2.5 inch strainers (B00428M7OI)
  • Ziploc disposable plastic dishes 8oz (top cut off)
  • Forceps
  • Bucket of ice


  • ImageXpress Nano set to 37°C (for quantifying L3 emergence)
  • 37°C CO2 incubator
  • Repeating 12-channel p1250 pipette with 1250 μL tips


  1. This assay should be performed at least 14 days after blood-feeding with microfilaria.

  2. Fill as many 96-well plates as necessary (3 plates is usually enough for 700 fed mosquitoes) with 200 μL warm RPMI.

  3. Remove cages and cartons of infected mosquitoes from the incubator and transfer to the screend room.

  4. Use a rubber bulb and glass tube to carefully remove all dead mosquitoes from the cartons. Place dead mosquitoes in the freezer overnight before discarding.

  5. Cold anesthetize the mosquitoes by putting the cartons at 4° for 3-5 minutes.

  6. Transfer knocked down mosquitoes into a glass Petri dish with a piece of filter paper on a bucket of ice. Then transfer the mosquitoes from the filter paper to a tea strainer and place the strainer in a dish filled with 70% ethanol (mosquitoes will sink). Soak for 2 min.

  7. Dip the strainer with mosquitoes in a new dished filled with dH2O. Gently shake the strainer, then remove it and dry the outside. Discard and replace the water, and soak the strainer with mosquitoes in the fresh water (mosquitoes should float).

  8. Use forceps to pluck floating mosquitoes (if any mosquitoes sink, don't use them) by the legs and plunge them head first into a well of RPMI. Fill the entire plate before moving to the next plate.

  9. After transferring all the mosquitoes, incubate the plate in the 37°C CO2 incubator for 1 hr.

  10. Before imaging, fill each well to the top with RPMI.

  11. Image each well on the ImageXpress at 2X.

  12. If desired, separate the head from the carcass and transfer the carcass to a new well. Gently disturb each tissue.

  13. Repeat steps 9-11.