Detergent-resistant membrane isolation and cholesterol measurement

Detergent-resistant membrane isolation and cholesterol measurement

Authors/Affiliations

Ayaka Ito, Xin Rong and Peter Tontonoz

Department of Pathology and Laboratory Medicine, Department of Biological Chemistry, and Molecular Biology Institute, David Geffen School of Medicine, University of California, Los Angeles, CA, USA

Correspondence to Peter Tontonoz: PTontonoz@mednet.ucla.edu

Abstract 

Detergent-resistant microdomains or lipid rafts are cholesterol- and sphingolipid-enriched membrane microdomains that are considered to function as membrane signaling platforms. Lipid composition in raft is critical determinant for membrane dynamics and downstream signaling. Signaling mediated by lipid rafts have been shown to play a key role in cytokine production and lymphocyte activation. Raft signaling is also associated with the pathogenesis of infectious, cardiovascular and neurological diseases. Here we present a protocol to isolate lipid rafts from primary murine bone marrow derived macrophages and quantify cholesterol abundance in isolated lipid rafts. 

Keywords

Lipid rafts, Cholesterol, Membrane

Materials and Reagents

1. Primary bone marrow macrophages 
2. Phosphate-buffered saline (PBS, see Recipes)
3. TNE buffer (see Recipes)
4. Protease inhibitor (Sigma P0044)
5. Phenylmethylsulfonyl fluoride (PMSF, see Recipes)
6. Triton X-100
7. OptiPrep (60% iodixanol, Progen Biotechnik, catalog number: 1030061)
8. Amplex Red Cholesterol Assay Kit (Invitrogen, catalog number: A12216)
9. BCA Protein Assay Kit (Pierce, catalog number: 23227)
10. Flottilin-1 antibody (Cell Signaling, catalog number: 3253)
11. Transferrin receptor antibody (Clone number H68.4; Invitrogen, catalog number 13-6800)

Equipment

1. Pipettes (P200, P1000)
2. Micro Centrifuge
3. 1 ml Open-top thickwall polycarbonate tube (Beckman Coulter, catalog number: 343778)
4. Ultracentrifuge (Beckman Coulter, Optima MAX-XP)
5. Fixed-angle rotor (Beckman Coulter, TLA-120.2)
6. Western blot equipment 

Procedure

Note: All following steps are performed at 4 °C or on ice if not stated otherwise. 


A. Detergent-resistant membrane (DRM) isolation

1. Culture macrophages at 3 x 10⁶ cells/dish in 10 cm dish at 37 °C in CO₂ incubator.
2. Wash cells with 5 ml PBS.
3. Wash with 5 ml TNE buffer.
4. Scrape and collect cells by adding 1 ml TNE buffer and transfer cell suspension into a 1.5 ml tube.
5. Centrifuge the cells for 5 min at 380 x G.
6. Suspend cell pellet in 110 μl TNE buffer with protease inhibitors and 200 mM PMSF, and homogenize with a 25-gauge needle (25 strokes).
7. Centrifuge the cells for 5 min at 1,000 x G to remove nucleus.
8. Transfer 100 μl cell homogenate to a new 1.5 ml tube and add 11 μl 10% Triton X-100.
9. Gently invert the sample to mix and incubate for 30 min on ice.
10. Adjust sample composition to 40% iodixanol by mixing (9) with 200 μl OptiPrep (60% iodixanol).
11. Transfer the 300 μl mixture from (10) to ultracentrifuge tube.
12. Overlay 600 μl 30% iodixanol onto (11).
13. Overlay 100 μl TNE buffer onto (12).
14. Centrifuge for 2 hr at 259,000 x G with the slowest acceleration and no deceleration.
15. Take 4 layers from top evenly (250 μl each, 4 fraction).
16. Analyze all fractions by Western Blot for protein markers.
17. Measure cholesterol concentration by directly using each fraction and Amplex Red Cholesterol Assay Kit, and normalized to protein concentration measured by BCA Protein Assay Kit.

Expected Results

DRM and associated proteins including Flottilin-1, a DRM marker, should be enriched in the second fraction from the top. While, soluble protein including transferrin, a membrane protein, and cell debris should be enriched in the third and fourth fractions from the top. An example of Western blot using flottilin-1 and transferrin to identify DRM has been published (Ito et al., 2015). 

Recipe

1. Phosphate-buffered saline

       140 mM NaCl

       2.7 mM KCl

2. TNE buffer

       50 mM Tris-HCl

       150 mM NaCl

       2 mM EDTA

       Adjust pH to 7.5

3. PMSF
       Dissolve 1.74 mg PMSF in 1 ml DMSO to make 100 mM PMSF. This can be stored at -20 °C.

Acknowledgments

The extraction method is adapted from Lingwood and Simons (2007).

Competing interests

The authors declare that no competing interests exist.

References

1. Ito, A., Hong, C., Rong, X., Zhu, X., Tarling, E. J., Hedde P. N., Gratton, E., Parks, J., and Tontonoz P. (2015). LXRs link metabolism to inflammation through Abca1-dependent regulation of membrane composition and TLR signaling. eLife 4: e08009.
2. Lingwood, D. and Simons, K. (2007). Detergent resistance as a tool in membrane research. Nat Protoc 2 (9): 2159-2165.

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