Skip to content

Ficoll Mononuclear Cells Isolation

Created by Ravishankar Madhu

Last Updated: 2023-07-10

Description

This protocol describes how to isolate WBCs (specifically mononuclear cells) via a Ficoll Density gradient. While this procedure takes longer than using Hemolytic Buffer, the added benefit is that granulocytes are depleted. Since granulocytes contribute greatly to the starting product of CD34+ enrichments, removing them before hand can reduce the input. Additionally, while thawing cells, granulocytes have a tendency to burst - leading to poor recovery of the cell product.

Things to Note before starting

With just reading / watching videos of a Ficoll Spin, the protocol might seem challenging to accomplish. However, on actual use - the procedure becomes much more clear. However, when you have the ability to use a Hemolytic Buffer Isolation, I would recommend doing so.

Reagents

  • Peripheral Blood, Bone Marrow, Chord Blood, or Apheresis Product.
  • Ficoll or Lympho Prep (https://www.stemcell.com/products/lymphoprep.html) - HAS TO HAVE DENISITY OF 1.077 g/mL
  • Hemolytic Buffer

Procedure

  1. Add 15mL of LymphoPrep / Ficoll into the bottom of a 50mL Tube.
  2. Using a Serological Pipette, gently add your input (PB /BM/Chord Blood/ or Apheresis) by dropping it slowly against the side of the tube. Make sure to not disrupt the interface between the LymphoPrep and your input. 1
  3. Reduce the deceleration on your centrifuge to 0.2
  4. Spin samples at 800 x g for 20 minutes. Due to the deceleration being set to 0 - this will end up taking ~45 minutes.
  5. After the spin you should have samples that look like this:

![[PeripheralBloodFicollSpin.jpg]]

The photo above is from a PB sample that was spun down using fritted 50mL tubes. You can see an upper yellow layer and a reddish/orange layer underneath. What we want to collect is the "Buffy Coat" that occurs at the interface between the Yellow and Red Layer.

![[BoneMarrowFicollSpin.jpg]]

In this photo above, you see BM samples after a Ficoll Spin. Unlike the PB sample, there is no clear yellow layer - this is because BM does not contribute a lot of serum. However, there are still two distinct layers. A orange layer at the top and a darker more red orangish layer in the middle. Again, you can see the thin white "Buffy Coat" of the cells we are trying to attain.

  1. Aspirate off the top layer of liquid, being careful not to disrupt the "buffy coat". 3
  2. Using a 10mL Serological Pipette, pick up as much of the "Buffy Coat" as possible. 4
  3. Transfer the "Buffy Coat" into a new 50mL tube.
  4. Pour 50mL of Hemolytic Buffer. Mix by inverting the tube.
  5. Incubate for 2-3 minutes.
  6. Spin at 800 x g for 5 minutes (MAKE SURE TO INCREASE DECELERATION TO NORMAL).
  7. aspirate off the supernatant, a small pellet should be visible at the bottom.
  8. Resuspend pellet in Hemolytic Buffer and bring total volume to 50 mL with Hemolytic Buffer.
  9. Incubate for 2-3 minutes.
  10. Spin at 800 x g for 5 minutes.
  11. Aspirate off the supernatant - a small pellet should be visible at the bottom.
  12. Resuspend the pellet in an appropriate volume and count via the Countess.

Next Steps

Most of the time, these cells are enriched for CD34 if there are enough WBCs for an input. Otherwise, the cells are stained with either the RM007b - NHP HSC Panel or RM007a - NHP Lineage Panel. Finally, these cells can also be frozen down.

Appendix

Recipe's for Buffers

Hemolytic Buffer

This reagent is used to lyse the RBCs from a sample of PB or BM to isolate only the WBCs. This should be stored in either the cold room, or in the TC Room Fridge if being used.

  • 32.1 g of Ammonium Chloride
  • 4.03 g of Sodium Bicarbonate
  • 0.8 mL of 500mM EDTA
  • 4L of Water
  • Sterilized by filtering with a 0.22um Vacuum Filter

The recipe can be scaled down depending on the volume of Hemolytic Buffer you need. Most commonly, this recipe is made in 1L (Divide everything by 4) or in 500mL (Divide everything by 8).

Footnotes

  1. This is considered to be the most complicated part of the protocol and the part that causes people to fear this technique. If you are having difficulties, try using a Fritted 50mL tube (catalog number: A2055-10EA) to create a separation between the LymphoPrep and your input ↩

  2. Usually, centrifuge acceleration and deceleration are kept at the maximum value ↩

  3. To avoid disrupting the buffy coat, you can leave a few mLs of the top layer present rather than aspirating right to the buffy coat. ↩

  4. Use the lowest suction setting while picking up the buffy coat and try to have the pipette just above the buffy coat to ensure you are not picking up any of the middle layer. ↩