Skip to content

Microfilariae Purification by Filtration

PD-10 Column Filtration

Materials

  • PD-10 Desalting Column (VWR, cat# 95017-001)
  • RPMI-1640 (Sigma, #R8758)
  • 50 mL conical tubes
  • 1M sodium azide

Gravity Filtration Protocol

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

  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 and either pour off the column storage if the column is new or allow the liquid to flow through the column if it has been used previously. If the column is new, cut the sealed end of the column at the notch.

  4. Equilibrate the column by adding 5 mL RPMI-1640 to the top of the column and allow the liquid to completely enter the packed bed. Repeat the equilibration 5 times or until ~25 mL of RPMI-1640 has been passed through the column. If the flow-through contains sodium azide, it must be collected and added to the sodium azide waste container in the fume hood.

  5. Add up to 1.5 million microfilariae suspended in 2.5 mL of media to the center of the column and allow it to enter the packed bed completely. Discard the flow-through.

  6. Elute the microfilariae in 5 mL aliquots of RPMI-1640 and check the flow-through for microfilariae. It will likely take at least 6 fractions (~30 mL) before the microfilariae begin to elute. Continue to elute in 5 mL aliquots until no more microfilariae are eluting.

  7. In a separate 50 mL conical tube, add 25 μL of 1M sodium azide to 25 mL RPMI-1640. This solution will be used to store the column so it can be reused in the future.

  8. 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 to the column in 5 mL aliquots. When 20 mL of the storage solution has flowed through the column, add the remaining 5 mL of storage solution and cap the column.

  9. Return the capped column to the 50 mL conical used for storing the column and add a tally mark to the tube to indicate the number of times the column has been used. Then store the column at 4°C for future use.

Note: PD-10 columns can be used up to 10 times using the gravity protocol if stored properly at 4°C with 0.01% sodium azide. Spinning the column in a centrifuge is not encouraged as it will pack the resin bed and inhibit the reusable capabilities of the column.

Filtration from Blood

Materials

  • RPMI-1640 (pre-warmed to 37°C)
  • 5 µm filter (Millipore Sigma, #TMTP02500)
  • Millipore reusable syringe filter (Sigma-Aldrich, #Z268429)
  • 10-20 mL syringe
  • PBS or water

Protocol

  1. Assemble the filtration unit by placing a 5 µm filter in the reusable syringe filter unit and then add a syringe with the plunger removed on the filter unit.

  2. Mix the microfiaremic blood 10-15 times by inversion and then add 1-3 mLs of blood to the syringe.

  3. Add PBS or water to the syringe to fill the remaining volume of the syringe and then add the plunger to the unit.

  4. Gently push the fluid through the filter unit. Do not push air through the filter unit or the microfilariae will dry on the filter membrane.

  5. Disconnect the syringe from the filter unit and remove the plunger. Add the syringe back to the filter unit and then fill the syringe with PBS or water to wash the microfilariae collected on the filter within the unit. Push the fluid through with the plunger.

  6. Quickly disassemble the filter unit and place the filter unit in RPMI-1640. Gentle vortexing can help release microfilariae from the filter unit although microfilariae loss should be expected.