|Figure 1. Image of lysosomal staining by Lyso-ID® Red dye. Increase in lysosome volume and number resulting from 200 µM verapamil.|
Here we describe the use of the EL406™ Combination Washer Dispenser to automatically aspirate media, wash cells and dispense reagents for the Lyso-ID® Red Detection kit, part of the CELLestial® Live Cell Analysis platform from Enzo Life Sciences.
Lyso-ID® Red dye is a fluorescent reagent that accumulates in lysosomes. An increase in signal is indicative of an increase in the number or size of cellular lysosomes and lysosome-derived vacuoles. In addition to the lysosomal specific dye, the assay also uses Hoechst 33342 nuclear stain. A decrease of 30% or greater of the Hoechst signal is indicative of generalized cytotoxicity.
H-mesothelioma (H-Meso) cells were seeded at 20,000 cells per well and allowed to attach overnight. The following morning the cells were treated with increasing doses of chloroquine, verapamil or thiostrepton. After an 18-hour exposure to the drugs the cells were washed and Lyso-ID® Red and Hoechst 33342 dyes were added using the EL406 Washer Dispenser. After 30 minute incubation, excess dye was removed by washing 3 times with wash solution, followed by a final addition of 80 µL of wash solution. The fluorescence was then determined using a Synergy Mx reader. Lyso-ID® Red dye (red fluorescence) was measured using an excitation of 540 nm and an emission of 680 nm, while Hoechst dye (blue fluorescence) was determined with an excitation of 340 nm and an emission of 480 (Figure 2).
|Figure 2. Schematic of the Automated Lyso-ID® Red Process Carried out by the EL406 Washer Dispenser and Synergy™ Mx Reader.|
Figure 3. Dose Dependent Drug Induced Increase in Lysosomal Content as Measured by Increase in Lyso-ID® Red Fluorescence
Figure 4. Response change in Lyso-ID® Red dye Fluorescence caused by thiostrepton and verapamil in two mesothelial cell lines
The Lyso-ID® Red kit in conjunction with the EL406 Combination Washer Dispenser provides an optimized workflow to investigate and screen compounds for phospholipidosis. The Lyso-ID® Red fluorescent assay kits provide a much easier way to screen for this potential problem than the gold standard method of electron microscopy. The ELx406 Washer Dispenser provides the liquid handling capabilities to automate many of the tedious fluid handling steps required of this assay. The EL406 instrument has the ability to quickly and reliably add and remove fluid from 96 or 384-well microplates containing live cells, replacing numerous manual pipetting tasks. In addition the device can dispense accurate amounts of Lyso-ID® Red reagent as needed.
1.Ikeda , K., M. Hirayama, Y. Hirota, E. Asa, J. Seki and Y. Tanaka (2008) Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes, BBRC, 377:268-274.
2.Lewis,J.H., F. Mullick, , K.G. Ishak, R.C. Ranard, B. Ragsdale, R.M. Perse, E.J. Rusnock, A. Wolke, S.B. Benjamin, L.B. Seeff, H.J. Zimmerman. (1990) Histopatholic Analysis of Suspected Amiodarone hepatoxicity. (Hum. Path. 21:59-67.
3.Donovick R, Pagano JF, Stout HA, Weinstein MJ (1955). Thiostrepton, a New Antibiotic I. In. vitro Studies Antibiotics Annual 3: 554–9.
4.Kwok, J. M-M., S.S. Myatt, C.M. Marson, R.C. Coombies, D. Constantinidou, and E. W-F. Lam (2008) Thiostrepton Selectively Targets Breast Cancer Cells through Inhibition of Forkhead box M1 Expression. Mol. Cancer Ther. 7(7):2022-2032
By, BioTek Instruments