Fig. 2: Isolation of Lipid Droplet associated Mitochondria (LDM) by differential centrifugation from rat liver. | Nature Communications

Fig. 2: Isolation of Lipid Droplet associated Mitochondria (LDM) by differential centrifugation from rat liver.

From: Lipid-droplet associated mitochondria promote fatty-acid oxidation through a distinct bioenergetic pattern in male Wistar rats

Fig. 2

a Schematic representation of the method to isolate LDM and Cytosolic Mitochondria (CM) from rat liver. The liver was dissected from Wistar rats and homogenized with a polytron homogenizer. Low-speed centrifugation separated the post-nuclear supernatant (PNS) from the pellet. PNS was layered with buffer B and subjected to centrifugation to separate the fat layer that contains crude LD and the supernatant containing CM. High-speed centrifugation stripped LDM from LD, and CM was pelleted from supernatant PNS fraction. Confocal images of the fat layer before (b) and after (c) stripping of LDMs. LDs were marked by BODIPY fluorescent dye and mitochondria by MitoTracker red dye. Note the preservation of LD-mitochondria contacts in the fat layer before stripping (b, zoom). Also, note that LDM has been effectively stripped of LD (c, MitoTracker) (scale bar, 20 μm). Experiment was performed with two biological replicates and multiple technical replicates were taken from each animal. d) Quantification of the LDs stained with MitoTracker in the fat layer before (b) and after stripping of LDMs (c) Data from multiple technical replicates from two independent biological replicates were taken and are presented as mean values ± SD. Significance was calculated by two tailed un-paired Student t-test. ****p < 0.0001. e LDM fraction stained with BODIPY and MitoTracker. Note that LDM is primarily free of LDs. f, g Comparison of BODIPY staining of the fat layer and LDM (scale bar, 20 μm). Experiment was performed with two biological replicates and numerous technical replicates were taken from each animal. g Quantification of the BODIPY intensity in LDs of fat layer (left panel of f) and LDMs (right panel of f). Data from multiple technical replicates from two independent biological replicates were taken and are presented as mean values ± SD. Significance was calculated by two tailed un-paired Student t-test. ****p < 0.0001.

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