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Lysosomal Trapping (Lysosomotropism) Assay

  • Lysosomes are essential for the degradation of old organelles and engulfed microbes and also play a role in programmed cell death.
  • Lysosomotropic agents e.g., chloroquine, accumulate preferentially in the lysosomes of cells in the body.
  • These agents tend to have both lipophilic or amphiphilic compounds with basic moieties. Once inside the acidic environment of the lysosome, the drug becomes protonated and trapped in the organelle.
  • Trapping of drugs in the lysosome may be one mechanism leading to drug-induced phospholipidosis for cationic amphiphilic drugs (CAD).
  • Cyprotex’s lysosomal trapping service is a cell based assay which uses high content screening to identify both lysosomotropism and cytotoxicity according to a recently published method.

Protocol

  Test System

High content screening using lysosomal and nuclear dyes (LysoTracker® Red DND-99 or LYSO-ID® Red)

  Cell Lines

HepG2, human liver carcinoma cell line. (Other cell lines or primary cells available on request, e.g., Retinal Pigmentation Epithelial Cells, iPSC-derived cardiomyocytes, iPSC-derived neurons, etc.)

  Test Compound Concentrations

Up to 8 point dose curve with top concentration based on cell loss or solubility limit

  Quality Controls

Vehicle control (DMSO)
Negative control = piroxicam
Positive control = chloroquine

  Compound Requirements

50 µL of 30 mM DMSO solution or equivalent amount in solid compound

  Data Delivery

Summary report
Minimum effective concentration (MEC) and AC50 value for each cell health parameter (cell loss, nuclear morphology, DNA fragmentation and lysosomotropism)
Graphical representation of data

 

Data

Lysosomal trapping (lysosomotropism)

Figure 1. Representative high content screening images for cells treated with (A) vehicle control (B) 50 µM chloroquine (a lysosomotropic agent) and (C) 150 µM piroxicam (a non-lysosomotropic agent) over a 4 hr period.  Drugs which are lysosomotropic such as chloroquine competitively inhibit uptake of the lysosomal dye into the lysosomes.

lysosomal trapping or lysosomotropism

 Figure 2. Graphical representation of lysosomal trapping data for chloroquine (a lysosomotropic agent) and piroxicam (a non-lysosomotropic agent). Chloroquine causes a concentration dependent decrease in lysosomal staining compared to vehicle control treated cells. No effect was observed for piroxicam. No cytotoxic effects were observed for either compound at the concentration range tested. Data represents mean of triplicate incubations ± standard deviation.