- Catalog number
- 9156
- Size / count
- 50 Tests
- Dry ice
- No
- Hazardous
- No
- Discontinued
- Yes
- Target
- Autophagy
- Label
- ICT
- Cell Type
- MCF-10A
- Type
- Cell Viabiity
- Detection Method
- Flow cytometry
- Wavelength
- 590 nm / 620 nm
- Assay Protocol
- 1. Prepare samples and controls in fresh cell culture medium (alternative buffers such as PBS may be used)., 2. Reconstitute Autophagy Probe, Red with 100 µL DMSO., 3. Dilute Autophagy Probe, Red 1:5 by adding 400 µL diH2O, PBS, or fresh cell culture medium., 4. Add diluted Autophagy Probe, Red to each sample at 1:50 spike (e.g. spike at 1:50 by adding 10 µL to 490 µL sample). The ideal staining concentration can vary based on cell line, application, etc., and should be determined by the end user. The recommended sample size is 0.5 mL., 5. Incubate approximately 1 hour., 6. Dilute 10X Cellular Assay Buffer 1:10 with diH2O., 7. Remove media and wash cells 3 times: add 1X Cellular Assay Buffer or fresh cell culture medium and spin cells., 8. Resuspend cell pellet in 1X Cellular Assay Buffer (typically 0.5 mL per sample)., 9. Analyze with a flow cytometer (using a green/yellow laser and appropriate filter). Autophagy Probe, Red excites at 590 nm and emits at 620 nm.
- Sample Type
- Cell culture
- Components
- Kit 9156: 50 tests, Autophagy Probe, Red, 1 vial, #6701, 10X Cellular Assay Buffer, 15 mL, #6694, Fixative, 6 mL, #636, Kit Manual
- Shipping Conditions
- Ships overnight (domestic), International Priority Shipping
- Storage Temperature
- Autophagy probe at ≤-20°C, other components at 2-8°C
- Notes
- Two Pack Kit
- Target Description
- 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.
