2016
DOI: 10.1007/978-1-4939-3756-1_19
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Isolation of Lysosomes from Mammalian Tissues and Cultured Cells

Abstract: Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fr… Show more

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Cited by 13 publications
(13 citation statements)
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“…For each sample, ten 15 cm × 2.5 cm ( D × H ) plates were used, and all steps were carried out with ice-cold reagents at 4°C. Lysosomal purification was performed following previously established protocol with minor modification ( 51 ). Cells were washed two times with PBS and carefully detached in PBS containing protease inhibitors (Sigma) (2 to 3 ml per plate) by using a cell lifter and transferred to tubes.…”
Section: Methodsmentioning
confidence: 99%
“…For each sample, ten 15 cm × 2.5 cm ( D × H ) plates were used, and all steps were carried out with ice-cold reagents at 4°C. Lysosomal purification was performed following previously established protocol with minor modification ( 51 ). Cells were washed two times with PBS and carefully detached in PBS containing protease inhibitors (Sigma) (2 to 3 ml per plate) by using a cell lifter and transferred to tubes.…”
Section: Methodsmentioning
confidence: 99%
“…[ 9–11 ] Moreover, it is a standard procedure to isolate lysosomes in sucrose gradient centrifuge technique. [ 4 ] The low conductivity buffer (9–10 µS cm −1 ) used herein consists of HEPES (2385 mg L −1 ), sucrose (80 700 mg L −1 ), and dextrose (4500 mg L −1 ) and is able to maintain 87% of the cells viable (for comparison phosphate‐buffered saline (PBS) buffer (13 mS cm −1 ) maintains 93.8% of the cells viable) for 1 h (Figure S5, Supporting Information) due to its physiological osmolality level. Trapping of a non‐fixed nucleus and mitochondrion in low‐conductivity buffer (9 µS cm −1 ) showed behavior similar to that of the fixed samples (see Figure S3, Supporting Information).…”
Section: Resultsmentioning
confidence: 99%
“…[ 2,3 ] Isolation of lysosomes followed by proteomic analysis has been used to identify new lysosomal membrane proteins. [ 4 ]…”
Section: Introductionmentioning
confidence: 99%
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“…Owing to their heterogeneity, low number, and marked fragility, the purification of lysosomes is a difficult task [41]. Lysosomes are capable of storing hydrophobic drugs with weak base characteristics by a mechanism that seems to be mediated by ABC proteins of the A subfamily [42], which produces a decrease in the concentration of the active antitumor drug in the cytoplasm [43].…”
Section: Subcellular Modelsmentioning
confidence: 99%