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PB or BM Lyse for WBC Isolation

Protocol by: Ravishankar Madhu

Last Updated: 2023-07-10

Description

This protocol describes how to collect Non-Human Primate (NHP) White Blood Cells (WBCs) from aliquots of NHP Peripheral Blood (PB) or NHP Bone Marrow (BM) using Hemolytic Buffer.

Reagents and Materials

Things to Note before starting

Safety Concerns

Always handle NHP PB & BM in a TC Hood while wearing a full-length lab coat and gloves to minimize any biohazard spills from animal material. Ensure you are up to date on your Hep B vaccinations in accordance with occupational health and EH&S regulations.

Estimated Timing

The protocol contains approximately 8 minutes of incubation and an additional 10 minutes (cumulative) in the centrifuge. The entire process, from PB and BM to WBC pellets, will take approximately 20-30 minutes. Keep this in mind when the TC room gets busy.

Procedure

  1. Remove the Hemolytic Buffer from the 4°C fridge and place it at room temperature inside the hood. 1
  2. Prepare and label 1 x 50 mL tube for every 10 mL of PB/BM received from the Primate Center.
  3. Aliquot 10mL of PB/BM into each 50mL Tube.
  4. Add Hemolytic Buffer into each tube until the volume reaches 50mL. Mix by inverting the tube.
  5. Incubate for 3-4 minutes. 2
  6. Spin solution at 800 x g for 5 minutes.
  7. Aspirate off the supernatant. A white cell pellet should appear at the bottom of the tube.
  8. Using a 10mL Serological Pipette, resuspend the pellet with 10mL of Hemolytic Buffer.
  9. Pour Hemolytic Buffer till volume reaches 50mL. Mix by inversion of the tube.
  10. Incubate for 3-4 minutes.
  11. Spin solution at 800 x g for 5 minutes.
  12. Aspirate off the supernatant. A more pronounced white pellet should be visible at the bottom of the tube.
  13. Resuspend the cell pellet in an appropriate volume and count with the Countess.

Next Steps

The WBCs can be utilized for a variety of different follow up steps. The most common us is to stain the cells for flow with either the Lineage or the HSC panel. Sometimes the WBCs are used in a CD34+ Enrichment. Any leftover cells are saved as a cell pellet.

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. A keen reader might ask why I perform this step, rather than just taking the Hemolytic Buffer out when I need to use it. I do not have any literature to support this step - just tradition from my time at the Fred Hutchinson. The working hypothesis was that, slightly thawed Hemolytic Buffer was more efficient in lysis. Your mileage may vary. ↩

  2. Some instructions call you to "incubate until solution darkens in color". I have found that the time it takes for the solution to darken in color varies drastically with little influence on the amount of lysing. Sometimes, the samples become "dark" in 30-40 seconds while other times it matches up with a 4 minute incubation. Due to the subjectivity in assessing when your "sample turns dark", I would recommend just setting a timer for 3minutes 30seconds. ↩