Rapid enrichment of mitochondria from mammalian cell cultures using digitonin

Abstract

We describe here a simple method to enrich mitochondrial fractions from mammalian cells for downstream analyses in the lab. Mitochondria purification involves cell lysis followed by separation of the organelles from the rest of the cellular components. Here, we use detergent to rupture the cell membrane of mammalian cells followed by differential centrifugation to enrich the organelles. Optimum conditions with respect to detergent concentration, time, sample size, and yield are discussed. The method's utility in downstream analyses and ease of processing multiple samples simultaneously is also described. All the reagents in this method can be assembled in-house, are economical, and are comparable, if not superior, to commercially available kits in terms of mitochondrial yield and integrity. • Rapid enrichment of mitochondria from mammalian cells using commonly available reagents. • Multiple samples can be processed simultaneously. • Works over a wide range of sample size (1 million to 100 million cells).

Utilisation of Oxford Nanopore sequencing to generate six complete gastropod mitochondrial genomes as part of a biodiversity curriculum

“…Given sufficient sequencing coverage, we found basecalling with high accuracy (hac) mode was able to produce mitogenomes with complete genes without premature stop codons. In addition to enriching for mitochondrial DNA experimentally 15 , 54 , the use of adaptive sampling recently developed in ONT allows preselected sequences to be enriched during the sequencing process 55 – 57 . This approach has proven successful in obtaining full mitogenomes of endangered animals by enriching host genomic DNA from fecal samples 56 .…”

Structural determinants of mitochondrial STAT3 targeting and function

“…In order to test the implication of the N-terminal domain of STAT3, we transfected HEK 293T cells engineered to lack endogenous STAT3 with a construct expressing either full length STAT3 (WT ST3) or a version of STAT3 lacking the first 132 amino acids (ΔNST3). 54 Mitochondria from transfected cells were purified either by combined treatment with digitonin and trypsin to remove peripherally associated proteins 43 or by digitonin/trypsin treatment followed by sucrose gradient purification to remove other intracellular organelles. 42 Cytosolic and mitochondrial extracts were analyzed by immunoblot using anti-STAT3 antibodies.…”

“…Where indicated, mitochondria were further purified prior to lysis by treatment with digitonin and trypsin, or by treatment with digitonin and trypsin followed by sucrose gradient purification, as previously described. 42 , 43 …”

miR-146a-5p mediates inflammation-induced β cell mitochondrial dysfunction and apoptosis

“…Mitochondria and cytoplasm from MIN6 cells were isolated using methods described previously (37). Briefly, 2×10 6 cells were seeded in a 10 cm culture dish and cultured for 3 days.…”