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Modulation of RBC cholesterol

RC Rima S Chakrabarti

Last updated date: Feb 1, 2021 Views: 823 Forks: 0

original An abbreviated version of this protocol was published in eLife in Feb, 2017
Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin
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Below is a protocol for modulation of RBC cholesterol as represented in the publication.

HPCD= (2-Hydroxypropyl)-β-cyclodextrin powder

PBSx buffer= PBS supplemented with 2% NCLPPS and 1mM EDTA

 

RBC Cholesterol Assay Protocol: Manipulate amount of cholesterol in RBCs

*Work on ice

Preparing human red blood cells:  

  1. Start with a 10mL human blood sample, freshly acquired (approved IRB protocol) from venipuncture (stored for up to 2 days at 4 degrees Celsius if needed. Centrifuge the sample @4000rpm for 8 min. Aspirate and discard supernatant. Resuspend the cell pellet in 1mL PBS buffer. Into 15mL centrifuge tube, pipette 300ul blood into 4.7mL PBSx buffer.
  2. Centrifuge @4000rpm for 8 min. 
  3. Aspirate supernatant
  4. Re-suspend ea pellet in 5mL PBSx.
  5. Centrifuge @4000rpm for 8 min.
  6. Repeat wash 1x (steps 4-5).
  7. Re-suspend pellets in 5mL PBSx.

Pre-treatment with cyclodextrin:

Sample 21605 TreatmentDiluted blood (ul)5% HPCD (ul)2.5mM Chol/CD (ul)PBSx buffer (ul)
1a0.5% HPCD40010001500
1b0.5% HPCD40010001500
2a0.25% HPCD4005001550
2b0.25% HPCD4005001550
3a0400001600
3b0400001600
4a25uM Chol/CD4000201580
4b25uM Chol/CD4000201580
5a50uM Chol/CD4000401560
5b50uM Chol/CD4000401560
  1. Incubate indicated samples for 30 minutes on rotator at room temperature.
  2. To end reaction, centrifuge samples at 4000rpm for 8 min. Aspirate supernatant. Re-suspend with 2mL PBSx. Repeat this wash 2x to remove cyclodextrin from RBCs.

 

 

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Chakrabarti, R(2021). Modulation of RBC cholesterol. Bio-protocol Preprint. bio-protocol.org/prep803.
  2. Chakrabarti, R. S., Ingham, S. A., Kozlitina, J., Gay, A., Cohen, J. C., Radhakrishnan, A. and Hobbs, H. H.(2017). Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin. eLife. DOI: 10.7554/eLife.23355

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