amplex red cholesterol fluorometric assay kit  (Thermo Fisher)

Journal: Journal of Lipid Research

Article Title: HBV-miR-3 induces hepatic cholesterol accumulation by targeting ABCA1 : Evidence for potential benefits of statin usage

doi: 10.1016/j.jlr.2025.100866

Figure Lengend Snippet: HBV-miR-3 expression leads to hepatocyte cholesterol build-up. A: Experimental design of the fluorometric cholesterol estimation assay performed for evaluating the expected increase in intracellular cholesterol post HBV-miR-3 expression in hepatocytes. B: Cellular cholesterol levels in cells (Huh7, HepG2) 48h after transfection with pHBV-miR-3, pcDNA3.1+, HBV 1.3x and microRNA-Scramble control (miR-SC). The cholesterol levels were normalised to respective total protein content. Bar graphs represent mean (±SD) of three independent experiments. C: Left panel: Hepatic lipid droplet accumulation visualised by BODIPY 493/503 staining in cells (Huh7, HepG2) 48 h post transfection with pHBV-miR-3 and pcDNA3.1+. The cells were counterstained with nuclear stain DAPI. Scale bar: 20 μM. Right panel: Quantitative analysis of BODIPY 493/503 fluorescence intensity was quantified in each micrograph and expressed as the mean ± standard errors (n = 6) in arbitrary units (a.u). ∗ P ≤ 0.05.

Article Snippet: The second aliquot was subjected to lipid extraction as described earlier ( ) followed by estimation of total cellular cholesterol content using the Amplex red cholesterol fluorometric assay kit (ThermoFisher Scientific).

Techniques: Expressing, Transfection, Control, Staining, Fluorescence

Journal: Biomedicines

Article Title: Utilizing Sphingomyelinase Sensitizing Liposomes in Imaging Intestinal Inflammation in Dextran Sulfate Sodium-Induced Murine Colitis

doi: 10.3390/biomedicines10020413

Figure Lengend Snippet: The inflammation targeting of the ICG loaded SM-liposomes was studied ex vivo from dextran sodium sulfate (DSS)-treated animals ( a ). The mice were treated with 4% DSS in drinking water for five days, and then regular water was given until the endpoint. Animals were sacrificed on days four, seven, and eight. Healthy non-treated animals were used as a control. The liposomes (125 μL 20 mM total lipid in PBS) were added via tail vein injection to the animals (n = 4 per disease group and control group). The mice were sacrificed, and the ICG fluorescence was analyzed from the proximal colon (dark column) and distal colon (white column) ex vivo 24 h after tail vein injection using a Night Owl Imager. Regions of Interest (ROIs) were drawn around two colon segments (proximal and distal colons) and were analyzed with the indiGO software. Y-axes represent Relative Fluorescence Intensity (RFI). Bars represent mean ± SEM. The targeting of the ICG- loaded SM-liposomes was verified by analyzing the ICG fluorescence from histological slices ( b ). The proximal (blue) and distal (grey) colon samples were collected and cut into 10 μm thin section slices. The fluorometric scanner was used to measure the ICG fluorescence from the thin section slices. Bars represent mean ± STDEV. Correlation between the aSMase activity ( x -axel) and targeted ICG fluorescence (y-axel) was analyzed ( c ). The ICG fluorescence was measured from proximal colon samples using a Night owl Imager; ROIs were drawn around proximal colon samples and were analyzed with the indiGO software. The homogenate samples were collected from the same proximal colon samples, and aSMase activity was analyzed using an Amplex Red acid sphingomyelinase assay kit. The Amplex fluorescence was standardized to the tissue weight (mg). All the tissue sample activities were analyzed simultaneously.

Article Snippet: Acid sphingomyelinase activity assay. aSMase activity was analyzed from collected cell supernatants or colon tissue homogenates using an Amplex Red reagent-based fluorometric kit (Thermo Fisher Scientific, Waltham, MA, USA) as previously described [ ].

Techniques: Liposomes, Ex Vivo, Control, Injection, Fluorescence, Software, Activity Assay