Autophagy Assay - Red Fluorescent

1. Prepare samples and controls., 2. Dilute 10X Cellular Assay Buffer 1:10 with diH2O., 3. Reconstitute FLICA with 50 µL DMSO., 4. Dilute FLICA 1:5 by adding 200 µL PBS., 5. Add diluted FLICA to each sample at 1:30-1:60 (e.g., spike at 1:30 by adding 10 µL to 290 µL sample)., 6. Incubate approximately 1 hour., 7. Remove media and wash cells 3 times: add 1X Cellular Wash Buffer and spin cells., 8. Resuspend cell pellet in 1X Cellular Wash Buffer., 9. If desired, label with additional stains, such as Hoechst 33342, DAPI, or an antibody., 10. If desired, fix cells., 11. Analyze with a fluorescence microscope or flow cytometer. FLICA 660 is excited at 660 nm and emits at 680-690 nm.

Autophagy is a conserved lysosomal recycling process by which cells break down their own components such as proteins, lipids, and carbohydrates. Autophagy plays a critical role in maintaining homeostasis by preventing the accumulation of damaged organelles by disassembling unnecessary or dysfunctional cells and cellular components. Autophagy occurs at low levels in the cell under normal conditions and can be rapidly upregulated during times of starvation or stress. Such degradation activities serve to provide nutrients (amino acids, nucleotides, fatty acids, etc.) and energy during periods of elevated bioenergetic demands. Another function of autophagy is to assist with the detection and destruction of intracellular pathogens (viruses, bacteria, and parasites). Dysregulation of autophagy has been associated with many disease states including cancer, infection, and degenerative diseases. Autophagy is a dynamic process typically divided into three stages (Figure 1). During stage one, cytoplasmic components targeted for degradation are sequestered within a double-membrane phagophore (also called the isolation membrane). This results in the formation of double-membrane vesicle called the autophagosome. During stage two, the autophagosome fuses with the lysosome to form the autophagolysosome or autolysosome. Degradation of the autophagosomal contents occurs during stage three. Our Autophagy Probe - Red Fluorescent enables researchers to detect and monitor the in vitro development of autophagy in living cells. Autophagy Probe - Red Fluorescent is a cell-permeant aliphatic molecule that fluoresces brightly when inserted in the lipid membranes of autophagosomes and autolysosomes. Autophagy Probe - Red Fluorescent can be readily detected by flow cytometry (Figure 2) with optimal excitation at 590 nm and peak emission at 620 nm. Autophagy Probe - Red Fluorescent is for research use only. Not for use in diagnostic procedures.