CYTO-ID® Autophagy detection kit

Protocol tips

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	Time Kinetics experiments are good to observe the optimal Cyto-ID staining (different time points between 6 to 24 hrs).

Protocol tips
Time Kinetics experiments are good to observe the optimal Cyto-ID staining (different time points between 6 to 24 hrs).

Publication protocol

Co-staining between LC3 and CYTO-ID
Living cells were stained with CYTO-ID (Enzo Life Sciences, Farmingdale, NY, USA) according to manufacturer's recommendations. Stained cells were resuspended in RPMI starvation media for 15 min at 4°C, allowing cells to attach to the poly-L-lysine coated microscopic slides. The cells were then fixed in 4% PFA, permeabilized with saponin, and stained with antibody against LC3 as described above. Images were acquired using a Confocal Laser Scanning microscope (LSM 710, Axio Observer, Carl Zeiss Inc.), equipped with 63 × 1.4 NA oil immersion objective, and the images were processed using the ZEN software.

Flow cytometry
All flow cytometry analyses were performed on a FACS Calibur instrument (BD Biosciences). For cell death analysis, cells were incubated with propidium iodide (PI) (20 μg/ml) for 10 min at 4°C. Quantification of autophagy was performed by using the CYTO-ID® Autophagy detection kit, according to the manufacturer's protocol. Data were analyzed using the CellQuest software (BD Biosciences).

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Manufacturer protocol

Download the product protocol from Enzo Life Sciences for CYTO-ID® Autophagy detection kit below.

Download manufacturer protocol

Videos

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