Quantitative analysis of autophagy in green fluorescent protein-light chain 3 transgenic murine keratinocytes

Abstract

Objective To explore a high-throughput method for quantitative analysis of autophago-somes. Methods Green fluorescent protein-light chain 3 (GFP-LC3) transgenic murine keratinocytes were randomly divided into 4 groups: control group receiving no treatment, starvation group subjected to starved culture, 20 J/cm2 ultraviolet A (UVA) group treated with 20 J/cm2 UVA radiation, and 40 J/cm2 UVA group treated with 40 J/cm2 UVA radiation. After 6-hour treatment, the cells were fixed, and images were acquired by confocal laser scanning microscopy. A macro was created by the ImageJ software to automatically quantify the GFP-LC3 puncta in the cells and the number of cells. Then, the level of autophagy was compared among different groups. Results By using the macro created by the ImageJ software, autophago-somes in the keratinocytes were successfully identified and quantified. Less than 0.6 second was needed for analyzing an image of 4.2 mega pixels in a test computer. The average number of autophagosomes in keratinocytes was significantly higher in the starvation group, 20-J/cm2 UVA group and 40-J/cm2 UVA group than in the control group whether with the treatment with pepstatin A (F = 20.05, P <0.05) or not (F = 5.01, P < 0.05) . This method could successfully differentiate the autophagy levels among the starvation group, UVA irradiation groups and control group. Conclusion A new high-throughput method, which can rapidly and accurately quantify GFP-LC3 puncta in cells, is established successfully to quantificationally detect autophagy. Key words: Autophagy; Keratinocytes; Green fluorescent proteins; Microscopy, confocal; Image processing, computer-assisted