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Fixation or RBCs for HbF Flow

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol will describe how to fix Peripheral Blood in preparation for Fetal Hemoglobin (HbF) flow.

Reagents and Materials

  • FACS Tubes
  • PBS
  • 0.05% Glutaraldehyde
  • 0.1% Triton
  • HbF antibody fluorochrome (usually in PE)
  • Parafilm

Things to Note before starting

Estimated Timing

There is about a dozen 5 minute spins causing this protocol to take ~1 hour to complete. After the samples are fixed, they are stable for 1 month, allowing you to run the HbF flow at your leisure. However I would recommend running the HbF flow the next day.

Making Buffers.

The 0.05% Glutaraldehyde is a working buffer that is actually made from a stock solution of 2% Glutaraldehyde. To create the 0.05% Glutaraldehyde, perform the following: - Add 1mL 2% Glutaraldehyde to 39mL of PBS

The 0.1% Triton is also a working buffer made from a stock solution of 1% Triton. To make the 0.1% Triton Solution, perform the following. - Add 1mL of 5mL 1% Triton to 45mL of PBS.

Procedure

Fixation or Red Blood Cells

  1. Add 100uL of fresh peripheral blood to 900uL of PBS. Mix by pipetting up and down.
  2. Label these tubes as the "Diluted PB pool" so that you have a supply that you can use again if you need to redo the flow.
  3. Transfer 100uL of the diluted pool into a new set of FACS tubes.
  4. Label these tubes as the "Samples to Fix".
  5. Add 3mL of 0.05% Glutaraldehyde to each of the tubes.
  6. Incubate at room temperature for 10-12 minutes. 1
  7. Spin down the samples at 800 x g for 5 minutes.
  8. Decant into the sink.
  9. Resuspend the sample by dragging the bottom of the FACS tubes against the grates of the TC Hood. 2
  10. Add 3mL of PBS. 3
  11. Centrifuge at 800 x g for 5 minutes.
  12. Decant into sink.
  13. Resuspend the sample by dragging the bottom of the FACS tubes against the grates of the TC Hood. 2
  14. **Repeat steps 10-13.
  15. Resuspend the pellet in 3mL of 0.1% Triton.
  16. Incubate at room temperature for 3-5 minutes. 4
  17. Spin down the samples at 800 x g for 5 minutes.
  18. Decant into the sink.
  19. Resuspend the sample by dragging the bottom of the FACS tubes against the grates of the TC Hood. 2
  20. **Repeat steps 10-13 Two Times.
  21. Seal the tubes with parafilm and store in the 4C Fridge until the samples are ready to be run for flow.

Staining for HbF Flow

  1. Add 10uL of fixed cells to 90uL of PBS into new FACS tubes. Mix by pipetting up and down.
  2. Add 5uL of HbF antibody.
  3. Incubate for 15-20 minutes at room temperature, while wrapped in aluminum foil.
  4. Wash samples with PBS.
  5. Spin down at 800 x g for 5 minutes. Decant into the sink without disrupting the pellet at the bottom.
  6. Run the samples on the Canto. Make sure to include 1 unstained control sample.

Next Steps

After flow is run and analyzed the samples can be tossed from the fridge. Explaining the HbF analysis is beyond the scope of this protocol. Speak to Stefan to learn how to analyze HbF Flow.

Appendix

Footnotes

  1. DO NOT INCUBATE SAMPLES FOR MORE THAN 12 MINUTES ↩

  2. You can also use the white FACS tube rack with the numerous vertical poles. This works well to resuspend the pellet after decantation. ↩↩↩

  3. Use the PBS Bottle to add the PBS as if you were washing cells for flow. ↩

  4. DO NOT INCUBATE SAMPLES FOR MORE THAN 5 MINUTES. ↩