Quantitative analysis of autophagy in green fluorescent protein-light chain 3 transgenic murine keratinocytes / 中华皮肤科杂志

ABSTRACT

Objective To explore a high-throughput method for quantitative analysis of autophagosomes.Methods Green fluorescent protein-light chain 3(GFP-LC3)transgenic murine keratinocytes were randomly divided into 4 groupscontrol 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.