Dnmt1KO and Dnmt1fl/fl peritoneal macrophages were seeded in 30 mm aperture laser confocal dish. Cells were treated with LPS at 100 ng/ml for 24 h at 37 °C. The cells were then washed with PBS for three times and incubated in phenol red-free RPMI 1640 medium containing 5 µmol/L NBD-cholesterol (Cayman, 13220, USA) for 4 h at 37 °C. Cell images were captured with Leica TCS-SP8 and Leica TCS-SP8 DIVE.
Nbd cholesterol
Manufactured by Cayman Chemical
Sourced in United States
NBD-cholesterol is a fluorescent cholesterol analogue. It is used as a tool compound for studying cholesterol trafficking and distribution within cells.
Automatically generated - may contain errors
Lab products found in correlation
Cholesterol and triglyceride assay kits, by Randox (2 mentions)
Apoa 1, by Merck Group (2 mentions)
Tcs sp8, by Leica (1 mentions)
Tcs sp8 dive, by Leica (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
α minimum essential medium, by Thermo Fisher Scientific (1 mentions)
Mip 2 elisa kit, by R&D Systems (1 mentions)
Human ldl, by Merck Group (1 mentions)
Mouse anti α tubulin antibody, by Merck Group (1 mentions)
Control small interfering rna sirna, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti abcg1, by Novus Biologicals (1 mentions)
Mouse anti abca1 antibody, by Abcam (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Fluorescent microscope, by Zeiss (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Zen 2009, by Zeiss (1 mentions)
Hoechst, by Cayman Chemical (1 mentions)
Turbofect, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Merck Group (1 mentions)
Mip 2, by R&D Systems (1 mentions)
8 protocols using nbd cholesterol
1
Cholesterol Uptake in Dnmt1 Deficient Macrophages
2
Macrophage Dysfunction and Lipid Dysregulation
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Recombinant mouse erythropoietin (rhEPO), macrophage colony stimulating factor (M-CSF), and macrophage inflammatory protein-2 (MIP-2) ELISA kits were obtained from R&D Systems (Minneapolis, MN, USA). Minimum essential medium α (MEMα) and RPMI 1640 medium were purchased from Gibco Life Technology (Karlsruhe, Germany). Goat anti-EPO, rabbit anti-EPOR, anti-βCR antibodies, control small interfering RNA (siRNA), and βCR siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-ABCA1 antibody was obtained from Abcam (Cambridge, MA, USA). Rabbit anti-ABCG1 was from Novus Biologicals (Littleton, CO, USA). Mouse anti-α-tubulin antibody, apolipoprotein AI (apoAI), high-density lipoprotein (HDL), 3,3-diaminobenzidine (DAB), bovine serum albumin (BSA), and human LDL were purchased from Sigma Chemical (St. Louis, MO, USA). NBD-cholesterol was from Cayman Chemical (Ann Arbor, MI, USA). Cholesterol and triglyceride assay kits were from Randox (Crumlin Co., Antrim, UK).
3
LDL and Cholesterol Uptake Assays
4
Macrophage Cholesterol Efflux Assay
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RAW264.7 macrophages were equilibrated with 1 μg/mL nitrobenzoxadiazole (NBD)‐cholesterol (13221; Cayman) in serum‐free RPMI1640 medium containing 0.2% BSA for 24 hours. The NBD–cholesterol‐loaded cells were rinsed and treated with 10 μg/mL exosomes or PBS for 2 hours. Then, 15 μg/mL apolipoprotein A1 (ApoA1) (A0722; Sigma‐Aldrich) or 50 μg/mL high‐density lipoprotein (HDL) (YB‐003; Yiyuanbiotech) was added into the culture medium to induce cholesterol efflux. NBD‐labeled cholesterol efflux was measured at an excitation of 470 nm and emission of 530 nm using a Microplate Reader (Tecan Infinite M200; LabX). ApoA1 or HDL‐mediated cholesterol efflux was expressed as the percentage of fluorescence in the medium to the total amount of fluorescence.
5
Macrophage Inflammatory Responses Assay
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Human LDL, HC030031, AITC, apoAI, HDL, Oil-red O, Gress's reagent, and mouse antibody for α-tubulin (clone DM1A, 1:10000) were from Sigma-Aldrich (St. Louis, MO, USA). Mouse antibody for TRPA1 (clone 6G8, 1:100 for immunohistochemistry or 1:1000 for western blot) was from Abnova (Taoyuan, Taiwan). Rabbit antibody for ABCG1 (E-20, sc-11150, 1:500) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse antibodies for ABCA1 (AB.H10, ab18180, 1:2000), F4/80 (CI:A3-1, ab6640, 1:100) and α-actin (1A4, ab7817, 1:100) were from Abcam (Cambridge, MA, USA). Macrophage colony stimulating factor (MCSF), TNF-α and ELISA kits for interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), IL-6 and macrophage I nflammatory protein-2 (MIP-2) were from R&D systems (Minneapolis, MN, USA). The ELISA kit for NF-κB activity was from Cayman Chemical (Ann Arbor, MI, USA). Dil-labeled oxLDL was from Biomedical Technologies (Stoughton, MA, USA). NBD-cholesterol was from Cayman Chemical (Ann Arbor, MI, USA). Fluo-8 Ca2+ assay kit was from AAT Bioquest (Sunnyvale, CA, USA). Cholesterol and triglyceride assay kits were from Randox (Crumlin, Co. Antrim, UK). TurboFect was from Fermentas (Glen Burnie, MD, USA).
6
Synthesis and Characterization of DBZ
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DBZ (purity: 99.6%) was synthesized by Dr Xiaohui Zheng at Northwest University (Xi'an, China). NBD cholesterol (Cayman), apoA1, high‐density lipoprotein (HDL) (ProSpec), atorvastatin (Pfizer Ltd.), lipopolysaccharide, PMA (phorbol 12‐myristate 13‐acetate), T0901317 (hereafter referred to as T1317), and GGPP (geranylgeranyl pyrophosphate) were purchased from Sigma‐Aldrich.
7
Cholesterol Efflux Assay in VSMCs
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VSMCs were incubated with ENVs or P-ENVs for 24 h and then incubated with 10 μmol/L Nitrobenzoxadiazolyl (NBD)-cholesterol (Cayman Chemical, Michigan, USA) for 4 h. After cholesterol loading, the NBD-cholesterol labeled VSMCs were washed with PBS twice and then incubated with apolipoprotein A-I (APOA-I, Sigma-Aldrich, St. Louis, USA; 15 μg/mL) for 4 h. The fluorescence-labeled cholesterol was released from VSMCs into the medium, and VSMCs were lysed at 37°C with 0.3 M NaOH solution for 15 min. The fluorescence intensity (FI) of the medium and lysate was measured at 472 nm excitation and 540 nm emission light using a microplate spectrophotometer. The efflux rate was calculated using the following equation: Efflux (%) = FI (efflux medium): [FI (efflux medium) + FI (cell lysate)] × 100.
8
Cellular Cholesterol Trafficking Assay
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Cholesterol uptake and export were determined using the Cholesterol Uptake Cell-Based Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). For cholesterol uptake assays, cells were incubated in media containing 10% LPDS for 24 h prior to treatment containing 20 ug/mL of a fluorescently tagged cholesterol (NBD cholesterol, Cayman Chemical Ann Arbor, MI, USA). Following 24 h treatment, cells were washed in PBS, assay buffer was added, and fluorescence was measured with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The cholesterol transport inhibitor U-18666A was used as a positive control. For cholesterol export assays, cells in 96-well plates were incubated with media containing 10% LPDS and 20 ug/mL NBD cholesterol for 48 h prior to treatment in media containing 10% FBS. Following 24 h treatment, the media containing exported NBD cholesterol was transferred to fresh wells and fluorescence measured as for uptake assays.
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