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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.

Saponin Hemolysis

Materials

  • 50 mL conical tubes
  • Sodium chloride
  • Saponin powder
  • Autoclaved water
  • 100-500 mL glass bottle

Equipment

  • Water bath (set to 37°C)
  • Scale

Protocol

  1. Prepare 0.85% sodium chloride, 0.2% saponin solution in autoclaved water. For example, in a glass bottle, combine 100mL autoclaved water with 850 mg sodium chloride and 200 mg saponin.

  2. In a 50 mL conical tube, make a 1:11 mixture of microfilaremic blood and saponin solution. For example, add 4 mL of blood to 44 mL saponin solution and mix by inversion.

  3. Place tube in 37°C water bath for 15 minutes to allow blood cells to lyse.

  4. Filter the solution, by following step 1 from the Filtration from Blood protocol (below), adding the lysis solution to the filter, and following steps 4-6 from the same protocol.

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. Filters can also be incubated in RPMI-1640 at 37°C for 1 hour for more passive separation.

Filtration from Blood (current)

Protocol from Zoetis adapted for the Zamanian Lab

Materials

  • ULTRA-WARE Glass 90 mm Microfiltration Assembly
  • Isopore 5 µm 90 mm membrane filters
  • Forceps
  • 1000 mL glass flask with vacuum attachment
  • Rubber hosing
  • Corning Cell Culture Flask with 0.2 µm Vented Cap
  • Corning 150 mL Storage Bottle
  • 10 cm cell culture dish
  • RPMI with P/S and FBS (complete media)
  • 2% Sodium Bicarbonate Solution or 1X PBS
  • Stopcock grease

Protocol

  1. Before filtration, create the RPMI media and 2% sodium bicarbonate solution.
  2. For the RPMI, add 25 mL of FBS and 5 mL of P/S to 500 mL of RPMI
  3. Keep this media in a 10 cm dish in the incubator at 37°C for at least 30 min before filtration.
  4. For the 2% sodium bicarbonate solution, add 20 g of solid sodium bicarbonate to 1000 mL of Millipore/DI water.
  5. Keep this media in the incubator at 37°C for at least 1 hr before filtration.

  6. If the filtered mf will be stained downstream, use 1X PBS for the filtration step and resuspend the mf in RPMI without FBS. The sodium bicarbonate solution may interfere with the success of the staining.

  7. Assemble the ULTRA-WARE glass 90 mm microfiltration assembly unit with the 1000 mL glass flask and rubber hosing. Add the isopore membrane filter in between the two pieces of ULTRA-WARE glassware. Connect the hose to a vacuum.

  8. When assembling be sure to add stopcock grease to the outer rim of the two pieces of glassware that connect to prevent leaking.
  9. The grease must be spread on the very edges because you want to minimize the amount of grease that can possibly get onto the isopore membrane filter.
  10. When adding the isopore membrane filter, use sterile forceps that have been cleaned with ethanol to take out of the package and handle the filter.
  11. Make sure the filter is centered, lays flat on the glassware, and has little bubbles or wrinkles.

  12. Do not touch the filter with anything other than the forceps.

  13. Once the glassware, filter, and hosing are set up, slowly turn on the vacuum and pour over 20-50 mL of sodium bicarbonate solution to wet the filter.

  14. Make a 1:1 dilution with the mf in blood and the sodium bicarbonate solution (or PBS) in the corning 150 mL storage bottle or a 50 mL conical tube depending on the amount of blood you have.

  15. Once the dilution is made and the membrane filter is wetted, pour over the dilution and slowly turn on the vacuum.

  16. Be sure to look carefully at the filter when the vacuum is on and that the filter does not become too dried out.

  17. Add more sodium bicarbonate solution or PBS to further rinse the blood out of the filter.

  18. When the filter looks free of blood, take apart the top glass piece of the assembly unit, remove the filter, and invert it into the 10 cm dish with the complete or incomplete RPMI media. Place in the 37°C incubator.

  19. When taking the filter off, it should not be completely dry but there should not be too much sodium bicarbonate left on the filter. Otherwise, when taking the filter off there will be leakage and loss of mf.

  20. In the dish, the media must be covering the filter completely.

  21. Let the filter sit in the media for about 5 minutes to allow the mf to settle and come off the filter.

  22. Transfer the mf in media to a corning cell culture flask with a 0.2 µm Vented Cap and store in the 37°C incubator until needed for assays.

  23. The mf are viable for up to 2 weeks after filtering

  24. To clean the assembly unit:

  25. Unclamp and remove the top piece of the unit.
  26. Use a paper towel to wipe off all stopcock grease.
  27. Remove the filter piece and run under DI water to clear as much blood from it
  28. Discard the blood in the collection flask in a bleach bucket and reattach the vacuum hose to it.
  29. Put the filter piece back onto the collection flask and run the vacuum to draw out the water still trapped in the filter. Do 2 more washes and vacuuming of the filter.
  30. Use the bleach squirt bottle to wash any other spattered blood that may be on the glassware into the bleach bucket.
  31. Glassware can be set on a paper towel next to the sink to air dry.