Monitoring Accessible Cholesterol Levels in Immune Cells

Abstract

Cholesterol is a critical lipid that is present at high concentrations in the plasma membranes of animal cells. Most of the membrane cholesterol is sequestered by other membrane lipids and the transmembrane domains of proteins. Cholesterol in excess of such sequestration forms a pool that is referred to as “accessible cholesterol.” This pool of cholesterol plays a crucial role in maintaining lipid homeostasis and in controlling cell growth. The accessible cholesterol pool can also be exploited by bacteria and viruses to promote infection and host immune responses rapidly lower levels of this pool to confer protection. We had previously developed a bacterial toxin sensor called ALOD4 to monitor and quantify accessible cholesterol in cultured cells. Here, we report the characterization of a modified version of ALOD4 that is specialized to detect and monitor accessible cholesterol levels in primary immune cells by flow cytometry analysis.

1. Introduction

Cholesterol is an essential lipid of animal cells whose levels are carefully controlled by complex regulatory mechanisms. Cholesterol is distributed unevenly among cellular membranes, with the highest concentration in plasma membranes (PMs) where it constitutes almost half of all lipids in that membrane [1]. Although present at such high concentrations, cholesterol can act as a specific signaling molecule, and small changes in cholesterol can regulate diverse signaling pathways. For example, in ciliary membranes, which are projections of the PM, cholesterol binding to a G-protein coupled receptor called Smoothened activates Hedgehog signaling and cell growth [2]. In another example, excess cholesterol that flows from the PM to the endoplasmic reticulum (ER) binds to an ER-embedded membrane protein called Scap to downregulate lipid production by inhibiting proteolytic activation of SREBPs [3, 4], which are transcription factors that control genes for lipid synthesis and uptake. A major challenge in membrane biology is to understand how a bulk lipid such as cholesterol can exert such specific signaling effects.

One answer to this problem lies in the organization of cholesterol in the PM. Most cholesterol in membranes is tied up in interactions with other membrane lipids such as sphingomyelin (SM) and membrane proteins, and it is only a tiny fraction of cholesterol that is in excess of the sequestration capacity of the membrane that participates in signaling reactions. This signaling pool of cholesterol has been referred to as “accessible” cholesterol [5]. Over the past decade, we have developed bacterial toxin-derived tools to monitor the accessible cholesterol pool in the PMs of cultured cells [6] and understand how it controls cell growth and lipid homeostasis. The most useful of these tools has been ALOD4, a fragment of the bacterial protein anthrolysin O [7, 8]. Using these tools, we and others have found that the accessible cholesterol pool can also be a vulnerability as several bacteria and viruses target it to promote infection, and we have uncovered a host immune response pathway that rapidly and selectively depletes this pool to confer protection [9–13]. The mechanism of this immune response involves rapid production in immune cells of an oxysterol called 25-hydroxycholesterol (25HC) that acts on multiple cholesterol regulatory pathways to deplete accessible cholesterol from PMs, thereby restricting microbial infection [9–13].

To extend the utility of ALOD4 to study primary immune cells that have much lower levels of accessible cholesterol than cultured cells, we need specialized modified forms of ALOD4 that retain specificity for this pool of cholesterol. Here, we provide a detailed description of one such form of ALOD4, which is covalently linked to biotin. This biotinylated ALOD4 serves as a specialized sensor that allows for efficient measurement of accessible cholesterol in mouse splenocytes by flow cytometry analysis.

2. Materials

2.1. Plasmid Transformation

1. pHis₆-Flag-ALOD4, designated as ALOD4, is a plasmid in the pRSET B expression vector that encodes, in sequential order from the NH₂-terminus, a 10-aa linker that includes a His₆ epitope tag (MRGSHHHHHH), a 23 aa-linker that includes a FLAG epitope tag (GMASMTGGQQMGRDYKDDDDKDP), and domain 4 (D4) of ALO (aa 404–512). The sole native cysteine in ALO’s D4 (C472) was mutated to alanine, and S404 was mutated to cysteine to allow for maleimide labeling at a position far from the cholesterol-binding site in ALOD4 (see Fig. 1a).

2. pHis₆-Flag-ALOD4(mut), designated as ALOD4(mut), is a version of the pHis₆-Flag-ALOD4 plasmid described above that contains six mutations (G501A, T502A, T503A, L504A, Y505A, and P506A) that disrupt cholesterol binding. ALOD4 and ALOD4(mut) plasmids have been described previously [14] and are available upon request.

3. BL21 (DE3) pLysS Escherichia coli competent cells (Invitrogen).

4. SOC medium (Invitrogen).

5. Water bath.

6. Microbiological incubator.

7. Incubator shaker.

8. LB-Agar-ampicillin plates: Add LB-Agar capsules (RPI) to the appropriate volume of distilled water, sterilize by autoclaving for 15 min at 121 °C, add ampicillin to a final concentration of 100 μg/mL, and while hot, pour 15–18 mL of LB-Agar-ampicillin into a 10-cm Petri dish.

Fig. 1.

Purification and characterization of ALOD4-Biotin. (a) Schematic of pHis₆-Flag-ALOD4 plasmid. The sole native cysteine in ALO’s D4 (C472) was mutated to alanine, and S404 was mutated to cysteine to allow for labeling with biotin-maleimide. (b) ALOD4 was purified and labeled with biotin, as described in the Methods section. Aliquots of unlabeled and biotin-labeled ALOD4 (2 μg each) were subjected to 15% SDS-PAGE, followed by Coomassie staining. (c) Gel filtration. Aliquots of ALOD4 (blue) and ALOD4-Biotin (red) were subjected to gel filtration chromatography on a Superdex^® 200 column. The maximum values for the absorbance at 280 nm for each protein were normalized to 1. (d) Mass spectrometry. Aliquots (0.5 μg each) of ALOD4 (blue) and ALOD4-Biotin (red) were subjected to mass spectrometry analysis. (e) Characterization of binding of ALOD4 and ALOD4-Biotin to CHO-K1 cells. On day 0, CHO-K1 cells were set up in medium A at a density of 6 × 10⁴ cells/well of a 48-well plate. On day 1, media were removed, and cells were washed twice with 500 μL of PBS, followed by the addition of 200 μL of medium A containing the indicated concentrations of ALOD4 or ALOD4-Biotin. After incubation at 37 °C for 1 h, cells were washed twice with 500 μL of PBS and harvested, and equal aliquots of cell lysates were subjected to immunoblot analysis. (f) Characterization of accessible cholesterol levels in CHO-K1 cells after 25HC treatment. On day 0, CHO-K1 cells were set up in medium A at a density of 6 × 10⁴ cells per well of a 48-well plate. On day 1, the medium was removed, and cells were washed twice with 500 μL of PBS, followed by the addition of 200 μL of medium A supplemented with the indicated concentrations of 25HC. After incubation at 37 °C for 4 h, the medium was removed and replaced with 200 μL of medium A supplemented with 3 μM of either ALOD4 or ALOD4-Biotin. After incubation at 37 °C for 30 min, cells were washed twice with 500 μL of PBS and harvested, and equal aliquots of cell lysates were subjected to immunoblot analysis

2.2. ALOD4 Expression, Purification, and Labeling

1. LB-ampicillin medium: Add one LB capsule (RPI) to 1 L of distilled water, sterilize by autoclaving for 15 min at 121 °C, and after cooling to room temperature, add ampicillin to a final concentration of 100 μg/mL.

2. 250-mL and 2-L flasks.

3. Incubator shaker.

4. Isopropyl β-D-thiogalactopyranoside (IPTG).

5. Cell counter.

6. Sorvall RC 3BP Plus centrifuge with a H-6000A swinging-bucket rotor for a 1-L bottle.

7. Eppendorf 5810R centrifuge with an A-4–81 rotor for 50-mL conical tubes (or equivalent).

8. Buffer A: 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 1 mM TCEP (Tris (2-carboxyethyl) phosphine).

9. Buffer B: Buffer A with 500 mM imidazole.

10. Buffer C: Buffer A with cOmplete^™, EDTA-free protease inhibitor cocktail tablets (Roche, one tablet per 50 mL of Buffer A).

11. Lysozyme from chicken egg white (Sigma-Aldrich).

12. Beaker.

13. Tip sonicator, Branson Digital Sonifier (S-250, Fisher Scientific).

14. Sorvall WX90 ultracentrifuge with a T865 rotor for 22-mL round-bottom tubes (or equivalent).

15. Nickel-nitrilotriacetic acid agarose (Ni-NTA) beads (QIAGEN).

16. 100-mL Dounce homogenizer (Kimble).

17. Sorvall MTX 150 Micro-ultracentrifuge with an AT-2 rotor (or equivalent).

18. Superdex^® 200 Increase 10/300 column (Cytiva).

19. ÄKTA pure FPLC system.

20. 15% SDS-PAGE gels.

21. 5× gel loading buffer 0.2 M Tris–HCl, pH 6.8, 10% (w/v) SDS, 10 mM β-mercaptoethanol, 20% (v/v) glycerol, and 0.05% (w/v) bromophenol blue.

22. Pierce^™ Bradford Plus Protein Assay Reagent (Thermo Scientific).

23. Quick Coomassie^® Stain (Anatrace).

24. Biotin-maleimide solution: Freshly prepare a solution of biotin-maleimide (Sigma-Aldrich) at a final concentration of 20 mM in DMSO.

25. 1.7-mL microcentrifuge tubes.

26. Rotator.

27. Dithiothreitol (DTT): Prepare a stock solution (1 M) in distilled water.

28. Gravity flow column (e.g., Poly-Prep^® Chromatography Column, Bio-Rad).

29. Amicon^® Ultra-4 10 kDa cutoff centrifugal filter.

30. NanoDrop^™ instrument or equivalent spectrometer.

31. Liquid N₂.

2.3. Measuring ALOD4-Biotin Binding to Cells

1. 24- and 48-well tissue culture plates (e.g., Corning).

2. Dulbecco’s Phosphate-Buffered Saline (PBS), 1× (e.g., Corning).

3. Medium A: Prepare a 1:1 mixture of Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F12 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin sulfate, and 5% (v/v) fetal calf serum (FCS).

4. 25-hydroxycholesterol (25HC, Avanti Polar Lipids).

5. Phenylmethanesulfonyl fluoride (PMSF).

6. Buffer D: 10 mM Tris–HCl, pH 6.8, 100 mM NaCl, 1% (w/v) SDS, 1 mM EDTA, 1 mM EGTA, 20 μg/mL PMSF, and cOmplete^™, EDTA-free protease inhibitor cocktail tablet (1 tablet per 20 mL).

7. Hydroxypropyl-beta cyclodextrin (HPCD).

8. Cholesterol/cyclodextrin complexes solution: Add cholesterol (in 100% ethanol) in small aliquots to a stirred solution of 5% (w/v) methyl-β-cyclodextrin (MCD) on a heating block (80 °C) until a clear solution was achieved. Lyophilize the solution. Reconstitute the complexes in pure water to reach a final sterol concentration of 2.5 mM.

9. Accumax^™ (Innovative Cell Technologies).

10. FACS buffer: PBS supplemented with 3% (v/v) FCS and 2 mM EDTA.

11. Streptavidin, Alexa Fluor^™ 594 conjugate (Invitrogen).

12. 16% (w/v) formaldehyde (e.g., Thermo Scientific).

13. Ghost Dye^™ Violet 450 (Tonbo Biosciences).

14. TruStain FcX^™ PLUS (anti-mouse CD16/32) antibody (BioLegend, 156604).

15. PE/Cyanine 7 anti-mouse CD45 antibody (BioLegend, 103114).

16. Flow cytometer (e.g., Stratedigm, S1000 flow cytometer with an A600 HTAS).

2.4. Isolation of Splenocytes from Mice

1. CO₂.

2. 70% (v/v) ethanol.

3. Mouse dissection tools (scissors and forceps).

4. PBS 1×.

5. 70-μm cell strainer (e.g., Falcon).

6. 50-mL conical tube.

7. 1-mL syringe.

8. RBC 10× (red blood cell) lysis buffer (e.g., BioLegend).

9. Trypan blue.

10. Hemocytometer.

11. 96-well v-shaped bottom plate (e.g., Corning).

3. Methods

3.1. Plasmid Transformation

1. Add 100 ng pHis₆-Flag-ALOD4 or pHis₆-Flag-ALOD4(mut) plasmid to 50 μL of BL21 (DE3) plysS cells, and incubate the mixture on ice for 30 min.

2. Heat-shock the cells by placing them in a 42 °C water bath for 30 s.

3. Immediately transfer the cells to ice for 2 min.

4. Recover the cells with 950 μL of SOC medium, and incubate them for 1 h at 37 °C.

5. Plate 100 μL of transformed and recovered cells on LB-Agar plates with ampicillin (100 μg/mL).

6. Incubate the plates for 16 h at 37 °C in a bacteriological incubator.

3.2. ALOD4 Expression

The protocol is the same for ALOD4 and ALOD4(mut).

1. Pick a single colony from the above plates, transfer it into a 250-mL flask containing 160 mL of LB-Amp, and incubate the flask at 37 °C in an incubator shaker at 225 rpm until OD₆₀₀ = 0.8–1.

2. Transfer 12 mL of the above starter culture to a 2-L flask containing 1 L of LB-Amp. Incubate the flasks at 37 °C in a shaker incubator at 225 rpm.

3. Once the OD₆₀₀ = 0.4–0.6, reduce the temperature to 18 °C, and continue shaking for 1 h at 225 rpm.

4. Add IPTG to a final concentration of 1 mM to induce ALOD4 expression, and continue shaking at 18 °C for 18 h.

5. Harvest the cells by centrifugation of 1 L cultures at 4600 × g for 10 min at 4 °C.

6. Resuspend pelleted cells from above in 1× PBS, and centrifuge at 3220 × g for 10 min at 4 °C.

7. Final cell pellets can be stored at −80 °C after flash freezing in liquid N₂.

3.3. ALOD4 Purification

The protocol is the same for ALOD4 and ALOD4(mut).

1. Resuspend the cell pellet from 1 L bacterial culture in 20 mL of Buffer C containing 1 mg/mL lysozyme (see Note 1).

2. Homogenize the cell suspension using a Dounce homogenizer.

3. Incubate the homogenized cell suspension at 4 °C for 1 h while stirring (or on a rotator).

4. After incubation, homogenize the cell suspension using a Dounce homogenizer.

5. Transfer the cell suspension into an empty beaker, and submerge the beaker in ice.

6. Lyse the lysozyme-disrupted cells using a tip sonicator: 35% amplitude, 3 min cycle (3s on, 3s off) followed by 6 min cool down.

7. Repeat the cycle two additional times.

8. Collect and centrifuge the cell lysate at 25,000 × g for 1 h in 22-mL round-bottom tubes.

9. Incubate the supernatant with 5 mL of Ni-NTA beads (pre-equilibrated in buffer C) on a rotator at 4 °C for 2 h (see Note 2).

10. Pour the mixture of supernatant plus Ni-NTA beads into a gravity flow column.

11. Wash the beads sequentially with 100 mL of Buffer C, 100 mL of Buffer C/Buffer B mix (95:5, v/v, 25 mM imidazole final concentration), and 100 mL of Buffer C/Buffer B mix (90:10, v/v, 50 mM imidazole final concentration).

12. Elute bound protein with 30 mL of Buffer C/Buffer B mix (60:40, v/v, 300 mM imidazole final concentration) into 3 mL fractions.

13. Use Bradford protein reagent (according to the manufacturer’s guidelines) to detect and pool protein-rich fractions.

14. Centrifuge eluted protein in 1-mL tubes at 100,000 × g for 1 h at 4 °C to remove aggregated protein.

15. Load 2 mL at a time onto a Superdex^® 200 column equilibrated in Buffer A (flow rate of 0.5 mL/min), and collect fractions using an ÄKTA pure FPLC system.

16. Determine protein concentration with a NanoDrop^™ instrument or equivalent. Typically, yields are high enough that further concentration is not necessary.

17. Collect protein-rich fractions, dilute as needed to a final 2 mg/mL concentration, and store at 4 °C (see Note 3). SDS-PAGE analysis with Coomassie staining in Fig. 1b (lane 1) shows the homogeneity of purified ALOD4. Figure 1c shows an example of the gel filtration profile of ALOD4 (blue curve).

3.4. Biotin Labeling of ALOD4

1. Combine 40 nanomoles (640 μg) of purified ALOD4 with 200 nanomoles of biotin-maleimide in a final volume of 1 mL of Buffer C in 1.7-mL microcentrifuge tubes (see Note 4).

2. Place tubes on a rotator at 4 °C for 20 h.

3. Quench labeling reactions by the addition of 10 mM DTT.

4. Add 200 μL of Ni-NTA beads equilibrated in Buffer A to the reaction mixture, and place it on a rotator at 4 °C for 1 h.

5. Load the mixture onto a gravity chromatography column.

6. Wash the beads with 10 mL of Buffer C, followed by 10 mL of Buffer C/Buffer B mix (90:10, v/v).

7. Elute bound ALOD4-Biotin using Buffer C/Buffer B mix (60:40, v/v) into a single 1 mL fraction.

8. Centrifuge eluted protein in 1-mL tubes at 20,000 × g for 1 h at 4 °C to remove aggregated protein.

9. Load labeled protein at a 0.5 mL/min flow rate onto a Superdex^® 200 column equilibrated with Buffer A using an ÄKTA pure FPLC system.

10. Pool protein-rich fractions, and concentrate using Amicon^® Ultra-4 10 kDa cutoff centrifugal filter to a final volume of 500 μL.

11. Measure the concentration of labeled protein using a NanoDrop^™ instrument. SDS-PAGE analysis with Coomassie staining in Fig. 1b (lane 2) and Fig. 2a shows the homogeneity of biotin-labeled ALOD4. Figure 1c (red curve) shows an example of the gel filtration profile of ALOD4-Biotin and its similarity to that of unlabeled ALOD4 (blue curve). Mass spectrometry analysis indicates ~100% labeling of ALOD4 with biotin (Fig. 1d).

Fig. 2.

Comparison of biochemical properties of ALOD4-Biotin and ALOD4(mut)-Biotin. (a) ALOD4 and ALOD4 (mut) were purified and labeled with biotin as described in the Methods section. Aliquots (2 μg) were subjected to 15% SDS-PAGE, followed by Coomassie staining. (b) Characterization of binding of ALOD4-Biotin and ALOD4(mut)-Biotin to CHO-K1 cells. On day 0, CHO-K1 cells were set up in medium A at a density of 6 × 10⁴ cells/well of a 48-well plate. On day 1, the medium was removed, and cells were washed twice with 500 μL of PBS followed by 200 μL of medium A containing the indicated concentrations of ALOD4-Biotin or ALOD4(mut)-Biotin. After incubation at 37 °C for 1 h, the cells were washed twice with 500 μL of PBS and harvested, and equal aliquots of cell lysates were subjected to immunoblot analysis. (c) Characterization of accessible cholesterol levels in CHO-K1 cells after 25HC treatment. On day 0, CHO-K1 cells were set up in medium A at a density of 6 × 10⁴ cells per well of a 48-well plate. On day 1, the medium was removed, and cells were washed twice with 500 μL of PBS, followed by the addition of 200 μL of medium A supplemented with the indicated concentrations of 25HC. After incubation at 37 °C for 4 h, the medium was removed and replaced with 200 μL of medium A supplemented with 3 μM of either ALOD4-Biotin or ALOD4(mut)-Biotin. After incubation at 37 °C for 30 min, the cells were washed twice with 500 μL of PBS and harvested, and equal aliquots of cell lysates were subjected to immunoblot analysis

3.5. Binding of ALOD4-Biotin to CHO-K1 Cells

Here, we describe how to treat cells with ALOD4, ALOD4-Biotin, or ALOD4(mut)-Biotin and obtain a cell lysate for further analyses. See Fig. 1e for an example of an assay that shows that ALOD4 and ALOD4-Biotin bind to the PMs of CHO-K1 cells at similar levels and that this binding leads to inhibition of cholesterol transport to ER, leading to a similar activation of SREBP2 cleavage. See Fig. 2b for an example of an assay that shows that ALOD4-Biotin, but not ALOD4(mut)-Biotin, binds to the PMs of CHO-K1 cells and activates SREBP2 cleavage. We have previously reported the mechanism by which oxysterol 25HC depletes accessible cholesterol from the PM [13]. See Fig. 1f for an example of an assay that shows that treatment with 25HC abolishes the binding of both ALOD4 and ALOD4-Biotin to the PMs of CHO-K1 cells. See Fig. 2c for another example of an assay that shows that treatment with 25HC abolishes the binding of ALOD4-Biotin to the PMs of CHO-K1 cells. As shown in Fig. 2c, no binding is observed for ALOD4(mut)-Biotin to cells under all conditions.

1. On day 0, set up CHO-K1 cells in medium A at 6 × 10⁴ cells/well in a 48-well plate.

2. On day 1, remove the cell culture medium from each well, and wash cells twice with 500 μL of PBS.

3. Add 200 μL of medium A containing indicated amounts of purified ALOD4, ALOD4-Biotin, or ALOD4(mut)-Biotin to each well.

4. After incubation for 1 h, wash each well twice with 500 μL of PBS.

5. Add 200 μL of Buffer D (cell lysis buffer) to each well, and place the 48-well plate on a shaker at room temperature for 10 min.

6. Mix equal aliquots of media and lysed cells with 5× loading buffer in microcentrifuge tubes, and incubate tubes at 95 °C for 10 min (see Note 5).

3.6. Measuring ALOD4-Biotin Binding to CHO-K1 Cells by Flow Cytometry

1. On day 0, set up CHO-K1 cells in medium A at 1.5 × 10⁵ cells/well in a 24-well plate.

2. On day 1, remove the medium from each well, and wash cells twice with 1 mL of PBS.

3. Add 500 μL of medium A without or with either 1% (w/v) HPCD or 100 μM cholesterol/cyclodextrin complexes.

4. Incubate the 24-well plates for 1 h at 37 °C.

5. After incubation, wash each well twice with 1 mL of PBS.

6. Add 200 μL of Accumax^™ to each well, and incubate at 37 °C for 5 min to dissociate the cells from the plate.

7. Transfer the detached cells to a 96-well v-shaped bottom plate, and pellet them by centrifugation at 800 × g for 5 min at room temperature. Carefully flick the plate to remove the supernatant.

8. Resuspend the cells in 100 μL of FACS buffer containing 1 μM ALOD4-Biotin or ALOD4(Mut)-Biotin.

9. Incubate 96-well v-shaped bottom plate for 30 min at room temperature.

10. Following the incubation, add 100 μL of FACS buffer, and pellet the cells by centrifugation at 800 × g for 5 min at room temperature.

11. Wash the cells twice with 200 μL of FACS buffer.

12. Pellet the cells by centrifugation at 800 × g for 5 min at room temperature, and remove the supernatant.

13. Resuspend the cells in 100 μL of FACS buffer containing streptavidin Alexa Fluor^™ 594 conjugate (at a 1:200 dilution).

14. Incubate for 30 min at 4 °C in the dark.

15. Following the incubation, add 100 μL of FACS buffer.

16. Pellet the cells by centrifugation at 500 × g at 4 °C for 5 min, and carefully remove the supernatant.

17. Wash the cells twice with 200 μL of FACS buffer per well (see Note 7).

18. Resuspend the cells in 100 μL of FACS buffer containing 1% (w/v) formaldehyde.

19. Incubate the cells for 20 min at 4 °C in the dark.

20. Pellet the cells by centrifugation at 800 × g at room temperature for 5 min, and carefully remove the supernatant.

21. Resuspend the cells in 150 μL of FACS buffer.

22. Cells are now ready for analysis on a flow cytometer. Stained cells can be stored at 4 °C in the dark for up to 5 days before analysis. See Fig. 3, panels a and b, for an example of flow cytometry analysis showing the binding of ALOD4-Biotin, but not ALOD4(mut)-Biotin, to CHO-K1 cells. The binding of ALOD4-Biotin is drastically reduced by HPCD treatment (which depletes PM cholesterol) and marginally increased by treatment with cholesterol/cyclodextrin complexes (which deliver cholesterol to the PM). Inasmuch as cultured cells have high amounts of accessible cholesterol, the age-dependent decline in ALOD4 activity does not significantly affect binding to CHO-K1 cells. In contrast, the age-dependent decline in ALOD4 activity results in a severe reduction of binding to primary cells such as mouse splenocytes (see below).

Fig. 3.

Flow cytometry analysis of accessible cholesterol levels in CHO-K1 cells and mouse splenocytes. (a and b) On day 0, CHO-K1 cells were set up in medium A at a density of 1.5 × 10⁵ cells/well of a 24-well plate. On day 1, the medium was removed, and the cells were washed twice with 1 mL of PBS, followed by one of three additions: (i) 500 μL of medium A, (ii) 500 μL of medium A supplemented with 1% (w/v) HPCD, or (iii) 500 μL of medium A supplemented with 100 μM cholesterol/cyclodextrin complexes. After incubation for 1 h, media were removed, and the cells were washed twice with 1 mL of PBS and incubated with 1 μM of either ALOD4-Biotin or ALOD4(mut)-Biotin as described in the Methods section. Bound biotinylated ALOD4 proteins were measured using Alexa Fluor^® 594-streptavidin by flow cytometry as described in the Methods section. (c–e) Splenocytes were isolated from mice, resuspended in PBS, and set up in a 96-well v-shaped bottom plate at a density of ~1 × 10⁶ cells/well. The plate was subjected to centrifugation at 500 × g for 5 min to pellet the cells, after which the supernatant was removed, and cells were resuspended in one of three buffers: (i) 100 μL of PBS, (ii) 100 μL of PBS supplemented with 1% (w/v) HPCD, or (iii) 100 μL of PBS supplemented with 100 μM cholesterol/cyclodextrin complexes. After incubation for 1 h, media were removed, and cells were washed twice with 200 μL of PBS and incubated with 1 μM of either ALOD4-Biotin or ALOD4(mut)-Biotin as described in the Methods section. Bound biotinylated ALOD4 proteins were measured using Alexa Fluor^® 594-streptavidin by flow cytometry as described in the Methods section

3.7. Isolation of Splenocytes from Mice

1. Euthanize the mouse by exposure to CO₂. After euthanization, move the mouse to the hood, and perform cervical dislocation.

2. Spray the mouse with 70% (v/v) ethanol.

3. Dissect the mouse longitudinally with scissors.

4. Move the intestines to the side with forceps to reveal the spleen next to the stomach.

5. Harvest the spleen, and remove fat from the spleen.

6. Place a 70-μm cell strainer into a 50-mL conical tube.

7. Place the spleen on the 70-μm cell strainer, and then, add 200 μL of PBS 1× onto the spleen.

8. Pulverize the spleen on the cell strainer with the flat end of the 1-mL syringe plunger until no chunks remain.

9. Wash the pulverized spleen on the cell strainer with 10 mL of cold PBS.

10. Pellet the cells in a 50-mL conical tube by centrifugation at 500 × g at 4 °C for 5 min.

11. Carefully aspirate the supernatant, and resuspend cells with 2 mL of RBC 1× lysis buffer.

12. Incubate the cells on ice for 2 min.

13. Following the incubation, add 18 mL of cold PBS.

14. Pellet the cells in a 50-mL conical tube by centrifugation at 500 × g at 4 °C for 5 min.

15. Resuspend the cells in 2 mL of cold PBS.

16. Mix the cells with trypan blue, and count the live cells on a hemocytometer.

3.8. Measuring ALOD4-Biotin Binding to Splenocytes by Flow Cytometry

1. Resuspend the cells in cold PBS (~1 × 10⁷ cells/mL), and set up cells for staining at 1 × 10⁶ cells per well in a 96-well v-shaped bottom plate.

2. Pellet splenocytes by centrifugation at 500 × g at 4 °C for 5 min, and carefully remove the supernatant.

3. Resuspend cells in 100 μL of PBS without or with either 1% (w/v) HPCD or 100 μM cholesterol/cyclodextrin complexes.

4. Incubate for 1 h at room temperature.

5. Following the incubation, add 100 μL of FACS buffer to all wells.

6. Pellet splenocytes by centrifugation at 500 × g at 4 °C for 5 min, and carefully remove the supernatant.

7. Wash the cells with 200 μL of cold PBS.

8. Pellet splenocytes by centrifugation at 500 × g at 4 °C for 5 min, and carefully remove the supernatant.

9. Resuspend the cells in 100 μL of PBS containing Ghost Dye^™ Violet 450 (at a 1:1000 dilution), which labels dead cells and provides a way to distinguish between live and dead cells.

10. Incubate for 30 min at 4 °C in the dark.

11. Following the incubation, add 100 μL of FACS buffer to quench the Ghost Dye^™ Violet 450.

12. Pellet splenocytes by centrifugation at 500 × g at 4 °C for 5 min, and carefully remove the supernatant.

13. Resuspend the cells in 100 μL of FACS buffer containing TruStain FcX^™ PLUS (anti-mouse CD16/32) antibody (at a 1:100 dilution) to prevent non-specific antibody binding to Fc receptors on immune cells.

14. Incubate for 30 min at 4 °C in the dark.

15. Following the incubation, add 100 μL of FACS buffer to all wells.

16. Pellet splenocytes by centrifugation at 500 × g at 4 °C for 5 min, and carefully remove supernatant.

17. Resuspend the cells in 100 μL of FACS buffer containing PE/Cyanine 7 anti-mouse CD45 antibody (at a 1:500 dilution), a marker for immune cells.

18. Incubate for 30 min at 4 °C in the dark.

19. Following the incubation, add 100 μL of FACS buffer to all wells.

20. Pellet splenocytes by centrifugation at 500 × g at 4 °C for 5 min, and carefully remove the supernatant.

21. Resuspend the cells in 100 μL of FACS buffer containing either ALOD4-Biotin or ALOD4(mut)-Biotin (see Note 6).

22. Incubate for 30 min at room temperature in the dark.

23. Following the incubation, add 100 μL of FACS buffer to all wells.

24. Pellet splenocytes by centrifugation at 500 × g at 4 °C for 5 min, and carefully remove the supernatant.

25. Wash the cells twice with 200 μL of FACS buffer.

26. Resuspend the cells in 100 μL of FACS buffer containing streptavidin Alexa Fluor^™ 594 conjugate (at a 1:200 dilution).

27. Incubate for 30 min at 4 °C in the dark.

28. Following the incubation, add 100 μL of FACS buffer to all wells.

29. Pellet the splenocytes by centrifugation at 500 × g at 4 °C for 5 min, and carefully remove the supernatant.

30. Wash cells twice with 200 μL of FACS buffer (see Note 7).

31. Resuspend cells in 100 μL of FACS buffer with 1% (w/v) formaldehyde.

32. Incubate for 20 min at 4 °C in the dark.

33. Pellet splenocytes by centrifugation at 500 × g at 4 °C for 5 min, and carefully remove the supernatant.

34. Resuspend the cells in 150 μL of FACS buffer.

35. Cells are now ready for analysis on a flow cytometer. Stained cells can be stored at 4 °C in the dark for up to 5 days before analysis. See Fig. 3, panels c to e, for an example of flow cytometry analysis of ALOD4-Biotin and ALOD4(mut)-Biotin binding to splenocytes. Note that ALOD4(Biotin) binding to splenocytes is about an order of magnitude lower than binding to CHO-K1 cells. Also, note the critical dependence of binding on the freshness of the ALOD4 probe (compare Fig. 3, panels c to d).

4. Notes

1. 1 L of His₆-Flag-ALOD4 bacterial culture usually yields 20–30 mg of ALOD4, and 6 L of His₆-Flag-ALOD4(mut) bacterial culture usually yields 5–10 mg of ALOD4(mut).

2. Overnight incubation with nickel beads increases protein binding and improves yield.

3. For optimal protein activity, purified proteins should be stored at 4 °C and used within 2 weeks. For best results, use proteins as soon as possible after purification (see Fig. 3, panels c and d, for an example of loss of protein activity over time).

4. Biotin labeling should be performed in 1.7-mL microcentrifuge tubes for maximum efficiency.

5. Use 10% SDS-PAGE for analyzing SREBP2 and 15% SDS-PAGE for analyzing ALOD4 and ALOD4(mut).

6. ALOD4-Biotin and ALOD4(mut)-Biotin labeling can be carried out on cells with or without fixation with 1% formaldehyde.

7. We recommend at least two washes during every step to reduce background during flow cytometry analysis.