The Cyto-IDTM Autophagy Detection kit (Enzo) was used in order to detect the autophagic flux. Cells were treated with 1 µM obatoclax or 0.1 µM paclitaxel and 1 µM obatoclax for 48 h. As positive controls, to block or induce autophagic flux, cells were incubated with 50 µM chloroquine or 800 nM rapamycin for 18 h, respectively. The cells were then stained with Cyto-IDTM Green dye and Hoechst 33342 using the Cyto-IDTM Autophagy Detection Kit (Enzo) according to the manufacturer’s protocol, visualized using an inverted fluorescence microscope (Zeiss, Oberkochen, Germany) and counted using CellQuest Pro software in a FACScan flow cytometer (BD Biosciences).
Cyto idtm autophagy detection kit
Manufactured by Enzo Life Sciences
Sourced in United States
The Cyto-IDTM Autophagy Detection kit is a fluorescence-based assay that can be used to quantify the level of autophagy in cells. The kit provides a fluorescent detection reagent that selectively labels autophagic vacuoles, allowing for the measurement of autophagy by flow cytometry, fluorescence microscopy, or fluorescence microplate readers.
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5 protocols using cyto idtm autophagy detection kit
1
Quantifying Autophagic Flux in Cells
2
Quantifying Autophagy in SW620 Cells
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Autophagy was monitored using the Cyto‐ID TM Autophagy Detection Kit (Enzo Life Sciences, France, ENZ‐51031‐K200) following the manufacturer's instructions. Briefly, for flow cytometric analysis, SW620 cells were harvested, washed with Assay Buffer, and stained with Cyto‐ID TM Green Detection Reagent at room temperature for 30 min in the dark. SW620 cell suspensions were then immediately analyzed by flow cytometry using Cell Quest software (FACScan, BD Biosciences).
3
Autophagy Detection using Cyto-ID Assay
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Cells were seeded on coverslips (SPL, Republic of Korea) and treated with vitexin and rapamycin (500 nm). Cells were fixed and stained using the Cyto-IDTM Autophagy Detection Kit (Enzo Life Sciences, Plymouth Meeting, PA) according to the manufacturer's instruction and counterstained with Hoechst 33342. Coverslips were mounted on glass slides with antifade (Invitrogen, USA) staining solution and analyzed using a fluorescence microscope. (Nikon Eclipse TS100 Epi-fluorescence microscope, Nikon Corp., Tokyo, Japan).
4
Autophagy Determination After Irradiation
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For autophagy determination 24 h after irradiation, we used the Cyto-IDTM Autophagy Detection kit (Enzo Life Sciences, Plymouth Meeting, PA, USA) according to the manufacturer’s instructions. Cells were washed in PBS (pH 7.2) and resuspended in 500 μL PBS containing Cyto-ID® Green Detection Reagent (0.1% v/v). Then, cells were incubated at 37 °C in the dark for 30 min and were resuspended in 200 μL PBS. The fluorescence emission of 10,000 cells was analyzed using a BD AccuriTM C6 cytometer and software (BD).
5
Chondrocyte Autophagy Induction by Clioquinol
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Chondrocyte autophagosome formation was detected by Cyto-IDTM autophagy detection kit (Enzo Life Sciences, Farmingdale, NY, USA), following the manufacturer’s recommendations. Articular cartilage cells of humans with OA were treated with or without 5 μM clioquinol for 48 h and then stained with a dual detection reagent for 30 min in the dark at 37°C. The green dot fluorescence (autophagosome) was observed under microscope.
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