Human podocytes differentiated for 13 days were labeled for 24 h at 37 °C in RPMI medium containing 2% FBS and [3H]-cholesterol (1 μCi/ml, American Radiolabeled Chemicals). Cells were then washed 3 times with PBS and incubated with RPMI supplemented with 0.2% fat free-BSA (Sigma) with or without compounds for 18-h at 37 °C. The compounds were used at the following concentrations: Cpd C (1 μM), Cpd A (1 μM, 5 μM) and Cpd G (1 μM, 5 μM, 10 μM). Following the 18-h incubation, human apoAI (Calbiochem) was added to the media, (20 μg/mL final concentration) and the cells were incubated for another 18 h at 37 °C. Aliquots of medium collected before (T = 0 h) and after (T = 18 h) the addition of apoAI were centrifuged at 12,000 × g for 5 min and the radioactivity in the supernatant was counted by liquid scintillation. Cells were then washed with PBS, lysed 0.1% SDS, 0.1M NaOH and the radioactivity in the lysates was quantified by liquid scintillation. apoAI-mediated cholesterol efflux, expressed as %, was calculated as the amount of label released to the medium after adding apoAI (the difference in radioactivity in the medium before and after adding ApoA1) divided by the amount of total label in each well (radioactivity released to the media plus radioactivity in the lysed cells).
3h cholesterol
Manufactured by American Radiolabeled Chemicals
Sourced in United States
[3H]-cholesterol is a radiolabeled form of cholesterol with tritium (3H) as the radioactive label. It is a useful tool for researchers studying cholesterol metabolism, transport, and related biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Q sepharose beads, by GE Healthcare (3 mentions)
Fat free bsa, by Merck Group (1 mentions)
Ezetimibe, by Merck Group (1 mentions)
Epoch plate reader, by Agilent Technologies (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Scintillation counter, by PerkinElmer (1 mentions)
Silica gel 60, by Avantor (1 mentions)
Sodium hydrosulfite, by Merck Group (1 mentions)
1 palmitoyl 2 oleoyl phosphatidylethanolamine, by Avanti Polar Lipids (1 mentions)
1 palmitoyl 2 oleoylphosphatidylcholine, by Avanti Polar Lipids (1 mentions)
Dextran sulfate, by Merck Group (1 mentions)
To901317, by Cayman Chemical (1 mentions)
Rpmi 1640, by Cayman Chemical (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Monoolein, by Merck Group (1 mentions)
Nhs activated sepharose 4 fast flow, by GE Healthcare (1 mentions)
Alexa fluor 488 goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw streptavidin, by LI COR (1 mentions)
Ezetimibe, by Santa Cruz Biotechnology (1 mentions)
8 protocols using 3h cholesterol
1
Cholesterol Efflux in Podocytes
2
Cholesterol Uptake Measurement Protocol
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Cholesterol-replenishing medium was prepared as follows. 3H-cholesterol (American Radiolabeled Chemicals) and MCD were mixed and combined in a glass vial, and solvents were evaporated under N2. The dried lipids were incorporated into medium B to a final concentration of 20 nM 3H-cholesterol and 375 μM MCD. For cholesterol uptake assay, on day 0, CRL-1601 cells that stably expressed hNPC1L1WT-Flag-GFP/hNPC1L1W347R-Flag-GFP/hNPC1L1L216A-Flag-GFP were maintained in medium A with 10% FCS at a density of 6 × 104 cells per well of 24-well plates. On day 1, the cells were treated with cholesterol-depleting medium with 0.1% dimethyl sulfoxide (DMSO; control) or 50 μM ezetimibe (Sigma-Aldrich) for 30 min at 37°C. The cells were then switched to cholesterol-replenishing medium with 0.1% DMSO (control) or 50 μM ezetimibe. The cells were washed three times with 0.25% (v/v) bovine serum albumin (Sigma-Aldrich)–added PBS and lysed in buffer B [20 mM Hepes (pH 8.0), 150 mM NaCl, and 1% n-dodecyl-β-D-maltopyranoside). The GFP intensity of lysates was measured using an Epoch plate reader (BioTek) at a wavelength of 488 nm. The cholesterol uptake of the same lysates was then measured by scintillation counter (PerkinElmer). The cholesterol uptake was normalized by GFP intensity.
3
Lipid Bilayer Membrane Characterization
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Porcine brain sphingomyelin (SM); 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-L-serine (POPS); cholesterol (CHOL) and 1-palmitoyl-2-6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl- phosphatidylcholine (C6-NBD-PC) were purchased from Avanti Polar Lipids (Alabaster, AL). [3H]-cholesterol was purchased from American Radiolabeled Chemicals, Inc (St. Louis, MO). Lipids were dissolved in chloroform and stored at −20°C. Concentrations of lipids were measured by dry weight. (2-hydroxypropyl)-α-cyclodextrin (HPαCD) average molecular weight 1180, 1 M 2,4,6-trinitrobenzenesulfonic acid in water (TNBS) and sodium hydrosulfite (sodium dithionite) were purchased from Sigma-Aldrich (St. Louis, MO). LW peptide (acetyl-K2W2L8AL8W2K2-amide) and pL4A18 peptide (acetyl-K2LA9LWLA9LK2-amide) were purchased from Anaspec (San Jose, CA) and used without further purification. High-performance thin-layer chromatography (HP-TLC) plates (Silica Gel 60) were purchased from VWR International (Batavia, IL).
4
Cholesterol Efflux Assay Protocol
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Cholesterol efflux assay was performed as previously described [27 (link)]. Briefly, THP-1 monocytes were differentiated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Merck) and incubated in serum-containing RPMI-1640 (Thermo Fisher Scientific, USA) with 0.6 μCi/ml [3H]-cholesterol (American Radiolabeled Chemicals Inc., USA) for 72 h. Cells were then incubated for 18 h in serum-free RPMI-1640 containing 4 μM LXR agonist TO-901317 (Cayman Chemical, USA). Cholesterol efflux was performed using 1.1% apoB-depleted plasma for 2 h. ApoB-depleted plasma was obtained after precipitation of apoB-containing lipoproteins with Dextran Sulfate (Merck, USA) as previously described [26 (link)].
5
In Vitro Sterol Binding Assay
6
In Vitro Sterol Binding Assay
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In vitro sterol binding was assessed using a previously described radioligand-binding assay (Im et al., 2005 (link), Choudhary and Schneiter, 2012 (link), Darwiche and Schneiter, 2017 (link)). Briefly, 100 pmol of purified protein in binding buffer (20 mM Tris, pH 7.5, 30 mM NaCl, 0.05% Triton X-100) were incubated with 0–400 pmol of [3H]-cholesterol (American Radiolabeled Chemicals Inc., St Louis, Missouri, USA) for 1 h at 30 °C. The protein was adsorbed to Q-Sepharose beads (GE healthcare) to remove unbound ligand, the beads were washed, and the radioligand was quantified by scintillation counting. For competition assays, 400 pmol of unlabeled cholesterol were included in the binding reaction, together with the indicated concentrations of [3H]-cholesterol. To determine non-specific binding, the ion exchange beads were incubated in the absence of added protein. At least two independent experiments were performed under each experimental condition and data are reported as the mean ± S.D. Calculation of the Kd value and curve fitting were performed using the statistical software Prism (GraphPad, La Jolla, CA, USA).
7
Cholesterol Trafficking Regulation Assay
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Cholesterol, U18666a, sodium taurocholate, mono-olein, oleic acid, phosphatidyl-choline (2-oleoyl-1-palmityl-sn-glycero-3-phosphocholine) were from Sigma-Aldrich (St. Louis, MO); ezetimibe and lysophosphatidylcholine (1-palmitoyl-sn-glycero-3-phosphocholine) were from Santa Cruz Biotechnology (Santa Cruz, CA); Ni-NTA resin was from Qiagen (Valencia, CA), NHS-activated Sepharose 4 Fast Flow and Q-Sepharose Fast Flow was from GE Healthcare Life Sciences. 3H-Cholesterol was from American Radiolabeled Chemicals (St. Louis, MO) and 3H-ezetimibe was from Merck. EZ-link Sulfo-NHS-SS-Biotin, SF900 III SFM insect cell medium, Freestyle 293 expression medium, Dulbecco’s modified Eagle’s medium (DMEM) and neutravidin agarose were from Life Technologies (Carlsbad, CA); lipoprotein-deficient serum was from KALEN Biomedical (Montgomery Village, Maryland). Chicken anti-GFP antibodies were from Life Technologies (used at 1:1000 for immunoblots). IRDye 680RD donkey anti-chicken and IRDye 800CW streptavidin were from LI-COR (Lincoln, NE) and used at 1:10,000 for immunoblots. Mouse anti-GFP antibody was from NeuroMab and Rabbit anti-LAMP1 antibody was obtained from Novus; both were used at 1:1000 for immunofluorescence. Alexa Fluor 488 Goat anti-mouse antibody and Alexa Fluor 568 Goat anti-rabbit antibody were obtained from Life Technologies and used at 1:2000 for immunofluorescence.
8
Radioligand Binding Assay for Cholesterol
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The radioligand binding assay was performed as described previously25 (link)47 (link). 100 pmol of purified protein in binding buffer (20 mM Tris, pH 7.5, 30 mM NaCl, 0.05% Triton X-100) was incubated with 0–400 pmol of [3H]-cholesterol (American Radiolabeled Chemicals Inc., St Louis, Missouri, USA) for 1 h at 30 °C. The protein was then separated from the unbound ligand by adsorption to Q-sepharose beads (GE healthcare, USA), beads were washed, and the radioligand was quantified by scintillation counting. The effect of 1,4-dioxane was determined by performing the in vitro assay in the presence of 1,4-dioxane (0–4%v/v). The effect of divalent cations on cholesterol binding was measured by performing the in vitro binding reaction in the presence of different concentrations of EDTA and magnesium chloride. At least two independent experiments were performed under each experimental condition and data is reported as the mean ± standard deviation. Calculation of the Kd value and curve fitting was performed using the statistical software GraphPad Prism, La Jolla, CA.
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