L3 filarial parasite chemotaxis assay.

<aside> ⚠️

Image paths corrected during migration. Two of the three images were linked as ../img/…, one directory level too high, so they did not display. The source also contains a commented-out reference to img1.png?s=100 — the query string would have broken that path too.

</aside>

Materials

• Agarose (molecular grade)
• 6 cm Petri dishes

Preparing chemotaxis plates

<aside> 💧

Make chemotaxis plates at least 48 hours in advance of the assay. Humidity plays a significant role in plate optimization for this assay.

</aside>

1. Make 0.8% (w/v) agarose plates. Microwave to dissolve, mixing intermittently. Pour ~10 mL of agarose into each 6 cm plate. Leave plates at room temperature with lids cracked for 24 hours.

2. Measure using the stencil and label the chemotaxis plate as shown below.

   

   Chemotaxis plate layout

Assay setup

1. Allow L3/L4 to soak in RPMI + P/S for ~30 minutes prior to chemotaxis assays, to re-sensitize to FBS.

2. Place 2 μL of DI H₂O in the M-area, then pick 8–10 L3s or L4s and place them in the DI H₂O in the M-area.

   

   Placing larvae in the M-area

3. After the L3s are placed, add 3 μL of test compound into the T-zone and 3 μL of DI H₂O into the C-zone.

4. Place plates in a 37°C incubator and monitor L3 migration every 10 minutes.

   

   Monitoring migration

5. Once the majority of L3 have begun to migrate, count the L3s in each zone or area. This includes counting the L3s that have not left the centre.

6. Run each condition in at least triplicate.

7. Store data using the template .csv files in the appropriate Box directory, and use existing scripts to calculate the chemotaxis index (CI).