Animal studies were performed in accordance with the University of Pennsylvania, institutional review board (IRB). LDLR-/-, APOBEC-1-/- double knockout (DKO) and LDLR-/-, APOBEC-1-/-, human ApoB100 transgenic (LAHB) mice were injected via tail vein for vector administration and serum collected by retro-orbital bleeds. At end of study animals were sacrificed and livers harvested for analysis. Vector were obtained from the Vector Core at the University of Pennsylvania and expressed cDNAs for hLDLR, hPCSK9 or hIDOL driven from a thyroxine binding globulin promoter (TBG). Mutations were introduced into wild type hLDLR using the Quick Change lightning kit (Agilent). Serum cholesterol levels were analyzed on a MIRA analyzer (Roche). Western blotting was done using precast mini gels (Invitrogen) and probed using a polyclonal hLDLR antibody. In vitro LDLR assay was performed by transiently transfecting HEK293 cells and pulsing the cells the following day with BODIPY labeled LDL (Invitrogen). Data were analyzed using one-way Analysis of Variance models with pair-wise group differences in mean cholesterol level assessed using Tukey's post-hoc tests.
Bodipy labeled ldl
Manufactured by Thermo Fisher Scientific
Sourced in United States
BODIPY-labeled LDL is a fluorescent-labeled low-density lipoprotein (LDL) product. The LDL is conjugated with a BODIPY fluorescent dye, allowing for the visualization and tracking of LDL in various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Precast mini gels, by Thermo Fisher Scientific (1 mentions)
Mira analyzer, by Roche (1 mentions)
Quickchange lightning kit, by Agilent Technologies (1 mentions)
Anti mouse cd16 32 fc block, by BD (1 mentions)
Fix perm buffer, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
3 protocols using bodipy labeled ldl
1
Humanized LDLR Mice for Cholesterol Analysis
2
Flow Cytometry Protocol for Cell Sorting
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Flow cytometry was performed as described (21 (link)) using anti-mouse CD16/32 (Fc Block; BD Biosciences) before staining with primary antibody or isotype controls. Cells were surface-stained, then fixed/permeabilized (Fix-Perm buffer, eBioscience) before intracellular staining. Antibodies are listed in Table 2 . Uptake of low-density lipoproteins was assessed by incubating PEC with BODIPY-labeled LDL (10 µg/ml, Invitrogen) (16 (link)). Data were acquired and analyzed as above. CD11b+Ly6Chi LyG- and CD11b+CD138+ Ly6G-cells were sorted using a FACSaria cell sorter and 40,000 cells/subset were lysed immediately for RNA extraction.
3
Cholesterol Uptake in HepG2 Cells
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Cholesterol liver uptake in cell was carried out according to the manufacturer's guide HepG2 cells were incubated with 15 μg/mL of BODIPY-labeled LDL (Invitrogen, Waltham, US) for 2 h at 37°C. Then, cell slides were then incubated with DAPI (Sigma-Aldrich, Missouri, US) for 5 min. Images were acquired using an Olympus microscope. Fluorescence density was analyzed using ImageJ software.
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