Microfilariae purification by filtration.

<aside> ⚠️

Two blood-filtration protocols are present, and a cross-reference between them is ambiguous. The source contains both "Filtration from Blood" and "Filtration from Blood (current)" — the first is presumably superseded but is never marked as such. The Saponin Hemolysis section then says to follow "step 1" and "steps 4–6" of "the Filtration from Blood protocol (below)", without saying which of the two. The step numbers match the older syringe-based protocol, so that is what is linked here, but this should be confirmed and the superseded version either deleted or clearly labelled.

</aside>

PD-10 column filtration

Materials

• PD-10 desalting column (VWR #95017-001)
• RPMI-1640 (Sigma #R8758)
• 50 mL conical tubes
• 1 M sodium azide

<aside> ☣️

A previously used column contains 0.01% sodium azide in the storage solution. Handle the column and storage solution appropriately, with gloves.

</aside>

Gravity filtration protocol

1. Remove the microfilariae species-specific PD-10 column from 4°C storage, or retrieve a new column.
2. Assemble the column in the adapter and place the setup in a 50 mL conical collection tube. The column and adapter can also be placed in a ring stand clamp with a flask underneath for fluid collection.
3. Remove the top cap. Either pour off the column storage if the column is new, or allow the liquid to flow through if it has been used previously. If the column is new, cut the sealed end at the notch.
4. Equilibrate the column by adding 5 mL RPMI-1640 to the top and allowing the liquid to completely enter the packed bed. Repeat 5 times, or until ~25 mL of RPMI-1640 has passed through.

<aside> ☣️

If the flow-through contains sodium azide, it must be collected and added to the sodium azide waste container in the fume hood.

</aside>

1. Add up to 1.5 million microfilariae suspended in 2.5 mL of media to the centre of the column and allow it to enter the packed bed completely. Discard the flow-through.
2. Elute the microfilariae in 5 mL aliquots of RPMI-1640, checking the flow-through for microfilariae. It will likely take at least 6 fractions (~30 mL) before the microfilariae begin to elute. Continue eluting in 5 mL aliquots until no more microfilariae are eluting.
3. In a separate 50 mL conical tube, add 25 μL of 1 M sodium azide to 25 mL RPMI-1640. This solution will be used to store the column so it can be reused.
4. When no more microfilariae are observed in the flow-through, wash the column and prepare it for storage by adding the RPMI-1640 containing sodium azide in 5 mL aliquots. When 20 mL of the storage solution has flowed through, add the remaining 5 mL and cap the column.
5. Return the capped column to the 50 mL conical used for storage and add a tally mark to the tube to indicate the number of times the column has been used. Store at 4°C for future use.