The Functional Analysis of Autophagy Using Lysosome-Specific Fluorescent Indicators

by Jinfang Liao, Zhenjun Diwu, Qin Zhao

Introduction

Autophagy is a general term for the degradation of cytoplasmic components within lysosomes. Lysosomes are cellular organelles which contain acid hydrolase enzymes, can digest damaged organelles, cell membranes and proteins, and engulfed viruses or bacterias (or other forms of waste) that invade a cell, and help to repair damage to the plasma membrane by serving as a membrane patch, sealing the wound.

The new LysoBrite™ dyes are a set of valuable tools for monitoring autophagy in live cells. Their signal intensity, photostability, cellular retention, as well as cytotoxicity of these lysosome indicators were evaluated in Hela and Jurkat cells with fluorescence microscope and flow cytometer. The functions of autophagy were analyzed using lysosome-selective LysoBrite™ dyes. These hydrophobic compounds easily permeate into intact cells, and are trapped in lysosomes. The LysoBrite™ fluorescence is significantly enhanced upon entering lysosomes, and they are well retained in cells with minimum cell cytotoxicity.

Methods

Cell cultures: Hela or Jurkat cells were plated in 96-well black wall/clear bottom costar plate at 37^oC, 5% CO₂ incubator for overnight.
Dye-loading: add equal volume of 1~2 µM of LysoBrite™ Orange, Red, Deep Red or NIR in 1X HBSS with 20 mM HEPES into each well of the cells. Incubate the cells at 37°C, 5% CO₂ incubator for 60 min. Cells were washed twice with buffer after dye loading.
Cell tracking: Hela or Jurkat cells are serially passaged at 1:1 ratio everyday after dye loading.
Analysis: Observe cell images under a fluorescence microscope (Olympus IX71), or analyze cells with a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA).

Fig. 1

The Emission spectra of LysoBrite™ Indicators

Results

Fig. 2

Images of Hela Cells stained with LysoBrite™ Red in a Costar 96- well Costar black wall/clear bottom plate. TRITC filter is used for imaging. Nuclei were stained with Hoechst 33342 (AAT Cat#17535).

Fig. 3

Images of Hela cells stained with A : LysoBrite™ Red (AAT Bioquest), B: LysoTracker^® Red (Invitrogen) in a Costar 96-well plate. The signals were compared at 0 and 120 seconds exposure time using an Olympus fluorescence microscope.

Fig. 4

Images of Hela cells stained with A: LysoBrite™ Red, B: LysoTracker^® Red in a Costar black wall/clear bottom 96-well plate. The images were compared at 3 cell passages (P1, P2 and P5) respectively using an Olympus fluorescence microscope (TRITC channel).

Fig. 5

Cell tracking assay with LysoBrite™ NIR (Ex/Em = 637/650 nm). Jurkat cells (~ 2x10⁶ cells/ml) were stained with LysoBrite™ NIR (2 µM) on Day 0. The cells were passed serially at 1:1 ratio for 23 days. Fluorescence intensity was measured with FACS Calibur flow cytometer (BD, San Jose, CA) in FL4 channel on the day after passage. Successive generations were represented by different colors.

Fig. 6

Phagocytosis of bacteria by Hela Cells. Three Hela cells were phagocytosing (engulfing) bacteria (red, labeled with LysoBrite™ Red analog). Blue and green colors were stained with Hoechst33342 (Blue) and Calcein AM (Green).

Fig. 7

Lysosome localization and motility is altered for starvation-induced Hela cells. A: Healthy untreated Hela cells. Lysosomes (Red) were dispersed widely throughout the cytosol in cells. B: Starved Hela Cells. The cells were starved for 24 hours (no serum), and lysosomes were aggregated in the perinuclear region. Nuclei were stained with Hoechst 33342.

Summary

LysoBrite™ dyes are characterized as a set of valuable tools for monitoring autophagy with the following benefits:

Minimal cytotoxicity: no cell toxicity were observed
Multiple wavelengths for multiplexing assays
Much brighter than LysoTracker^® dyes
Extraordinarily high photostability
Excellent cellular retention

Document: 02.0012.130116r1

Last updated Thu Aug 28 2025

The Functional Analysis of Autophagy Using Lysosome-Specific Fluorescent Indicators