Delipidation and LD induction were performed as described previously (Salo et al., 2016 (link)). Cells were delipidated by culturing in serum-free medium supplemented with 5% lipoprotein-deficient serum (Sigma) for 60 h. Nascent LD formation was induced by adding 0.2 mM OA (final concentration) for the indicated times.
Lipoprotein deficient serum
Manufactured by Merck Group
Sourced in United States
Lipoprotein-deficient serum is a laboratory reagent used in biochemical and cell culture research. It is a type of serum that has been processed to remove lipoproteins, such as cholesterol, triglycerides, and other lipid-containing molecules. This specialized serum is used as a cell culture supplement to study the effects of lipoproteins on cellular processes and metabolism.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fluvastatin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Mouse monoclonal anti actin antibody, by Merck Group (1 mentions)
Anti myc antibody, by OriGene (1 mentions)
Hyclone fcs, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
Pravastatin, by Merck Group (1 mentions)
Colorimetric assay, by Merck Group (1 mentions)
1 14c oleic acid, by PerkinElmer (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Blasticidin s, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Fujifilm (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Nh4cl, by Merck Group (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Fluoromount g with dapi, by Thermo Fisher Scientific (1 mentions)
12 protocols using lipoprotein deficient serum
1
Nascent Lipid Droplet Formation
2
Lassa Virus Infection and Inhibition
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Vero-E6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Inc., Grand Island, NY, USA) supplemented with 10% (vol/vol) fetal calf serum (FCS, HyClone; Thermo Scientific, Waltham, MA, USA) and penicillin-streptomycin (Life Technologies, Inc.). In some samples, sterol-depleted medium (lipoprotein-deficient serum; Sigma-Aldrich, St. Louis, MO, USA) was used instead of medium with FCS. Huh7 cells were propagated in DMEM with 10% (vol/vol) FCS and 1× nonessential amino acids (Life Technologies, Inc.). 25HC was from Sigma-Aldrich. LASV (Josiah strain) was from the CDC Viral Special Pathogens Branch reference collection; all work with LASV was done under BSL-4 conditions. The plasmid encoding LASV-GPC has been previously described (21 (link), 32 (link)). Plasmids encoding Myc-tagged CH25H and Myc-tagged IFITM3 were purchased from Origene (Rockville, MD, USA). The following antibodies were used in this study: mouse monoclonal antibody against LASV NP (703079-Lassa Josiah HMAF), mouse monoclonal antibody against LASV GP1 (52-74-7), monoclonal antibody against LASV GP2 (81001-52-2085-0006-BG12-alpha-LASVG2), and a mix of 5 monoclonal LASV antibodies for immunofluorescence (SPR628 anti-LASV 5-MAb mixture). The anti-Myc antibody was from Origene, and the mouse monoclonal antiactin antibody was from Sigma-Aldrich (St. Louis, MO, USA).
3
Quantifying LDL Uptake in Infected Endothelial Cells
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The binding of LDL to receptors on the surface of infected cells and non-infected controls was estimated using Bodipy-labelled LDL (Life Technologies). Therefore, E. bovis-infected BUVEC (15 days pi) were cultivated in FCS-free endothelial cell basal medium (PromoCell) supplemented with 10% lipoprotein deficient-serum (Sigma-Aldrich) for 36–48 h prior to medium supplementation with Bodipy-labelled LDL (10 μg/mL). The cells were incubated for 1 h at 4 °C followed by 4 h at 37 °C and also 3 h, 5 h for fluorescence microscopy. For qualitative analyses the cells were washed in PBS, fixed (4% paraformaldehyde, 10 min) and mounted in Prolong antifading mounting medium prior to fluorescence microscopy. For quantitative analyses, BUVEC were treated with 3% NaN3 (5 min, RT) to inhibit receptor recycling and washed with ice-cold PBS. Monolayers were detached by Accutase (Sigma-Aldrich) treatment (5 min, 37 °C), washed in PBS (400 × g, 5 min) and transferred to 5-mL FACS tubes containing 200 μL of PBS. The cells were processed in a FACS Calibur flow cytometer using the FL1-H channel.
4
Cholesterol Modulation Techniques in Cells
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Cholesterol starvation was performed on cells with 70–80% confluency. Pravastatin (20 µm ) (Sigma) was incubated with cells cultured in Lipoprotein Deficient Serum (Sigma) for 24 h before lysis. Cholesterol depletion was performed on starved, confluent cells. The cells were incubated for 30 min with 1% methyl‐β‐cyclodextrin (MβCD) in DMEM, after which subsequent incubations were done in DMEM with 20 µm Pravastatin. For cholesterol rescue experiments, cholesterol was added back by incubating the cells for 1 h with soluble cholesterol‐MβCD complexes (100 µm in DMEM supplemented with 40 µm Pravastatin) after either cholesterol starvation or depletion. When the assays were finished, the cells were lysed and subjected to immunoblotting. For cholesterol supplementation in cell growth both 2D or 3D, cholesterol‐MβCD complexes were added at the final concentration of 10ug mL−1 in the culture medium.
5
Culturing HL-1 Cardiomyocytes for Pterostilbene Treatment
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The mouse HL-1 cardiomyocyte cell line (SCC065) was obtained from Sigma-Aldrich (St. Louis, MO, USA) and cultured in the DMEM media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Rockford, IL, USA) for 24 h, and then it was transferred to DMEM medium supplemented with 10% lipoprotein deficient serum (Sigma-Aldrich); this mixture was maintained overnight at 37 °C in a humidified 5% CO2 incubator. After reaching ≥95%, the confluency cells were passaged. For treatment, the cells were incubated in a medium containing a vehicle (0.1% DMSO) or pterostilbene in a humidified incubator at 37 °C and 5% CO2 for 24 h.
6
Lipid Metabolism in Pancreatic Islets
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Thin layer chromatography (TLC): Islets (∼250) were cultured in RPMI 1640 media containing lipoprotein deficient serum (Sigma) for 16 h with 1 μCi of [1–14C] oleic acid (17 pmol; Perkin Elmer). Lipids solubilized in chloroform were resolved by TLC as described [27] . Total Acyl-Glycerols: Measured in primary islets, as previously described [28] (link). Briefly, batches of 130–200 freshly isolated primary islets were subject to chloroform:methanol (2:1 vol:vol) extraction, dried material resuspended in isopropanol, and acyl-glycerol content measured by colorimetric assay (Sigma).
7
Cell Cholesterol Regulation Assay
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Cholesterol, 25-HC, CHX, fluvastatin, lipoprotein-deficient serum, MG132, and NH4Cl were purchased from Sigma (St Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) was from Wako (Osaka, Japan). Blasticidin S was from Invitrogen (Carlsbad, CA, USA). SFaN and SFeN were from LKT Laboratories (St Paul, MN, USA).
8
Analyzing Lipid Accumulation in BMDMs
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CSF1-cultured BMDMs were plated on coverslips in 10% lipoprotein-deficient serum (Sigma-Aldrich) in RPMI overnight before being treated with 25 μg/ml oxidised low-density lipoprotein (oxLDL) (ThermoFisher Scientific) for 24 h. Cells were washed with PBS twice and stained with 5 μM BODIPY™ 493/503 (ThermoFisher Scientific) in PBS for 30 min at 37 °C. After two wash steps, cells were fixed in 4% paraformaldehyde (PFA) (ThermoFisher Scientific) for 10 min at room temperature and were mounted with Fluoromount-G with DAPI (ThermoFisher Scientific).
9
Luciferase Assays for Sterol Regulation
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Luciferase assays were done using a modified version of previously described protocols (Oeffner et al., 2009 (link); Zelenski et al., 1999 (link)). Briefly, cells were plated in DMEM:F12 supplemented with 5% lipoprotein‐deficient serum (Sigma‐Aldrich) and 20 μM sodium oleate (Sigma‐Aldrich) in a 96‐well plate at a density of 15,000 cells/well. On Day 1, cells were transfected with a 1:10 mixture of Renilla luciferase driven by a constitutive thymidine kinase promoter and firefly luciferase driven by a hamster HMG‐CoA synthase promoter containing a sterol regulatory element (SRE) (gift of Timothy Osborne, Addgene #60444). FuGENE HD (Promega) was used as the transfection reagent according to the manufacturer's protocol. Four hours posttransfection, the media was changed to sterol‐deficient media (DMEM:F12 with 5% lipoprotein deficient serum, 50 μM lithium mevalonate [Sigma‐Aldrich], and 50 μM compactin [Sigma‐Aldrich], or sterol‐enriched media with the addition of 10 μg/ml water‐soluble cholesterol [Sigma Aldrich] and 1 μg/ml 25‐hydroxycholesterol [Sigma‐Aldrich]). After 24 h, luciferase activity was measured using the Dual‐Luciferase Reporter Assay System (Promega) in a SpectraMax L microplate luminometer with reagent injectors (Molecular Devices).
10
Quantifying Endothelial oxLDL Uptake
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Images were captured using a Ziess LSM 880 confocal, Ziess 710 confocal or a Ziess Axiovert Fluorescent Microscope with a long-distance 40× lens.
oxLDL uptake was quantified by adding 1 µg/ml of fluorescent rhodamine labeled oxLDL (medium oxidized DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-oxLDL, Alfa Aesar) into the flow medium for the duration of the experiment (48 h). In all experiments involving oxLDL, FBS is substituted with 2% lipoprotein deficient serum (Sigma-Aldrich). After the cessation of flow, the cells were fixed and washed within the flow chamber: washed twice with PBS (with Ca/Mg), once with a 0.5 M NaCl 0.2 M acid wash buffer and twice with PBS. The intermediate wash step with the acid wash buffer ensures removal of any remaining extracellular DiI-oxLDL particles bound to the EC surface. Average cellular fluorescence was assessed using ImageJ by outlining 4–7 cells per image and 3–5 images per condition for each experiment. For experiments not involving flow, ECs were treated with 50 µg/ml DiI-oxLDL for 1 h, a dosage and time previously determined to induce EC stiffness15 (link).
oxLDL uptake was quantified by adding 1 µg/ml of fluorescent rhodamine labeled oxLDL (medium oxidized DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-oxLDL, Alfa Aesar) into the flow medium for the duration of the experiment (48 h). In all experiments involving oxLDL, FBS is substituted with 2% lipoprotein deficient serum (Sigma-Aldrich). After the cessation of flow, the cells were fixed and washed within the flow chamber: washed twice with PBS (with Ca/Mg), once with a 0.5 M NaCl 0.2 M acid wash buffer and twice with PBS. The intermediate wash step with the acid wash buffer ensures removal of any remaining extracellular DiI-oxLDL particles bound to the EC surface. Average cellular fluorescence was assessed using ImageJ by outlining 4–7 cells per image and 3–5 images per condition for each experiment. For experiments not involving flow, ECs were treated with 50 µg/ml DiI-oxLDL for 1 h, a dosage and time previously determined to induce EC stiffness15 (link).
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