Autophagy is a conserved lysosomal recycling process by which cells break down their own components such as proteins, lipids, and carbohydrates. The process plays an important role in maintaining homeostasis and destroying intracellular pathogens. In addition, autophagy can be upregulated in times of starvation or stress to provide additional nutrients for the cell. Dysregulation of autophagy has implications for cancer, infection, and degenerative diseases.
Autophagy is a three-stage process. First, cytoplasmic components targeted for degradation are sequestered, resulting in the formation of the autophagosome. Next, the autophagosome fuses with the lysosome to form the autophagolysosome or autolysosome. Finally, degradation of the autophagosomal contents occurs.
Our Autophagy Assay, Red (cat# KF17373) enables researchers to detect and monitor the in vitro development of autophagy in living cells. The Autophagy Probe is cell-permeant and fluoresces red when inserted in the lipid membranes of autophagosomes and autolysosomes. Results can be read using a flow cytometer.
Figure. Flow Cytometry Results. Autophagy Assay Kit, Red was used to assess the induction of autophagy in Jurkat cells. Cells were either untreated (Black) or treated with 0.5 μM Rapamycin (Orange), 10 μM Chloroquine (Blue), or both 0.5 μM Rapamycin and 10 μM Chloroquine (Red) for 18 hours. After staining with Autophagy Probe, Red for 60 minutes, cells were washed and analyzed by flow cytometry (BD LSRFortessa Special Order flow cytometer equipped with a green/yellow laser (561 nm excitation) and a 610/20 emission filter). An overlay of the histograms is shown on the right. A table displaying the median fluorescence signal, % negative, and % positive cells is shown below. Treatment with Rapamycin or chloroquine increased the fluorescence signal detected compared to the untreated control. Combined treatment of rapamycin and chloroquine further increased the fluorescence signal detected. Data courtesy of Dr. Kristi Strandberg (ICT 228:37-40).
We have many options for detecting apoptosis, necrosis, autophagy, and cytotoxicity in living cells.