Skip to Content
Merck
  • Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay.

Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay.

Autophagy (2015-06-27)
Sonja Aits, Jennifer Kricker, Bin Liu, Anne-Marie Ellegaard, Saara Hämälistö, Siri Tvingsholm, Elisabeth Corcelle-Termeau, Søren Høgh, Thomas Farkas, Anna Holm Jonassen, Irina Gromova, Monika Mortensen, Marja Jäättelä
ABSTRACT

Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ammonium-14N chloride, 99.99 atom % 14N, 15N-depleted, 99% (CP)
Sigma-Aldrich
8-Octanoyloxypyrene-1,3,6-trisulfonic acid trisodium salt, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Dimethyl sulfoxide, PCR Reagent
Sigma-Aldrich
Etoposide, synthetic, 95.0-105.0%, powder
Sigma-Aldrich
Hematoxylin, certified by the Biological Stain Commission
Sigma-Aldrich
Bromophenol Blue, titration: suitable
Sigma-Aldrich
Hematoxylin
Sigma-Aldrich
Glycerol, BioUltra, for molecular biology, anhydrous, ≥99.5% (GC)
Sigma-Aldrich
Phenol Red, ACS reagent
Sigma-Aldrich
Fluorescein, for fluorescence, free acid
Sigma-Aldrich
o-Xylene, anhydrous, 97%
Sigma-Aldrich
Dimethyl sulfoxide, BioUltra, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Propidium iodide solution
Sigma-Aldrich
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O
Sigma-Aldrich
Dimethyl sulfoxide, anhydrous, ≥99.9%
Sigma-Aldrich
Glycerin, meets USP testing specifications
Sigma-Aldrich
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 10% in H2O
Sigma-Aldrich
Dimethyl sulfoxide, sterile-filtered, BioPerformance Certified, meets EP, USP testing specifications, suitable for hybridoma
Sigma-Aldrich
Glycerol, BioXtra, ≥99% (GC)
Sigma-Aldrich
Glycerol, for molecular biology, ≥99.0%
Sigma-Aldrich
Terfenadine
Sigma-Aldrich
Thapsigargin, ≥98% (HPLC), solid film
Sigma-Aldrich
Triton X-100, BioXtra
Sigma-Aldrich
Dimethyl sulfoxide, for molecular biology
Sigma-Aldrich
Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
Sigma-Aldrich
Dimethyl sulfoxide, meets EP testing specifications, meets USP testing specifications
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Sigma-Aldrich
Phenol Red, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Glycerol, ≥99.5%