The Cyto-ID kit (ENZO Life Sciences, Lörrach, Germany) was used for quantifying autophagy. Cells were seeded in 6 cm dishes, being careful that cells were confluent when harvesting. The supernatant from each sample was transferred to a 15 mL tube, cells were rinsed with PBS and trypsinized with 1 mL trypsin-EDTA and taken up in 1 mL fresh medium, which was transferred to a 15 mL tube for centrifugation (1000 rpm, 5 min). The pellet was resuspended in 2 mL PBS, washed again in PBS and resuspended in 0.25 mL DMEM with 5% FBS without phenol red and 0.25 mL diluted Cyto-ID solution was added to each sample. After resuspension, the samples were incubated 30 min at 37 °C in the dark. After centrifugation (1500 rpm, 5 min), the supernatant was discarded and the pellet was resuspended in 1 mL assay buffer. Samples were centrifuged (1500 rpm, 5 min) and resuspended in 0.5 mL assay buffer and transferred into FACS tubes. FACS Canto was employed for the measurement. The data were analysed using the BD FACSDiva software.
Cyto id kit
Manufactured by Enzo Life Sciences
Sourced in Germany, United Kingdom
The Cyto-ID kit is a fluorescent dye-based product designed for the detection and quantification of autophagic vacuoles in living cells. The kit provides a selective staining of autophagic vacuoles, allowing for their visualization and analysis using fluorescence microscopy or flow cytometry.
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Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Lysotracker dnd 99, by Thermo Fisher Scientific (1 mentions)
Gp62 c, by Novus Biologicals (1 mentions)
M csf elisa, by R&D Systems (1 mentions)
Nb100 2220, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Pp242, by Merck Group (1 mentions)
Monodansylcadaverine mdc, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Imatinib, by LC Laboratories (1 mentions)
Dm irbe 2 epi fluorescence microscope, by Leica (1 mentions)
Anti β actin ab, by Merck Group (1 mentions)
Gs 700 imaging densitometer, by Bio-Rad (1 mentions)
Facscalibur, by BD (1 mentions)
7 protocols using cyto id kit
1
Quantifying Autophagy Using Cyto-ID Assay
2
Osteoclastogenesis Regulation by WDFY3
3
Autophagy Pathway Modulation in K562 Cells
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K562 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 μg/ml penicillin. The Cyto-ID kit was purchased from Enzo Life Sciences. PP242, chloroquine, bafilomycin A1 and monodansylcadaverine (MDC) were purchased from Sigma-Aldrich. Imatinib was purchased from LC Laboratories. Stock solutions were prepared as follows: Imatinib (10 mM) was dissolved in either sterile water or DMSO; PP242 (20 mM) was dissolved in DMSO; chloroquine (10 mM) was dissolved in sterile water; bafilomycin A1 (5 mM) was dissolved in DMSO.
4
Quantify Autophagosomal Content in Murine HSC
5
Quantifying Autophagy in Hematopoietic Stem Cells
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Autophagosomal content was quantified with the CytoID kit (Enzo Life Sciences, Exeter, UK). Murine BM was cultured in CytoID dye (1 : 3000) containing medium for 30 min according to the manufacturer's protocol. Cells were stained with surface antibodies to identify HSC (Lin−Sca1+cKit+CD150+CD48−) and LSK (Lin−Sca1+cKit+) populations and were analyzed using flow cytometry. Rapamycin and Wortmannin were used as internal controls.
6
Quantification of Cellular Uptake of SCO-Complexes
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HeLa pLuc/705 7 × 103 cells were seeded in a 96-well plate with glass bottom 24 h before treatment. Cells were incubated in serum-containing media with/without ligand for 1 h; then the fluorescent PF14/SCO(Alexa568) complexes (MR10, 100 nM SCO) were applied and incubated with cells for 24 h. Cells were then stained with Cyto-ID kit (Enzo Life Sciences) according to the manufacturer’s instruction. Imaging was performed using Leica DM/IRBE 2 epi-fluorescence microscope controlled by Micro-Manager19 with a 63 × 1.4 NA oil immersion objective. Images were analyzed using Fiji (ImageJ) software20 . Quantification was performed by acquiring a stack with 0.3 µm slices through the cell. The stack was filtered with the 3D mean filter using 2 × 2 × 2 setting. Cells were then manually marked as a region of interest (ROI) and the signal from the ROIs with the highest average intensity used. Cell-free ROIs next to the measured cell was used for background subtraction.
7
Autophagy Quantification in EA.hy926 Cells
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For the analysis of autophagy, EA.hy926 cell samples (untreated and exposed to TNF and/or etanercept) were lysed in lysis buffer. After SDS-PAGE, Western blot was performed using a rabbit antihuman LC3B and rabbit anti-human p62 antibodies (Abs) (Cell Signaling Technology). Peroxidase-conjugate goat anti-rabbit IgG (Bio-Rad) was used as a secondary Ab. To ensure the presence of equal amounts of protein, the membranes were reprobed with anti-β-actin Ab (Sigma-Aldrich). Quantification of protein expression was performed by densitometry analysis of the autoradiograms (GS-700 Imaging Densitometer, BioRad). The Cyto-ID kit (ENZO Life Sciences) was used for flow cytometry quantification of autophagy, by determining the percentage of autophagosome formation. At the end of experiments, cells were pelleted and then resuspended in DMEM without phenol red, supplemented with 5% FBS, and diluted Cyto-ID solution was added to each sample. After resuspension, the samples were incubated 30 min at 37°C in the dark. After centrifugation (1500 rpm, 5 min), samples were resuspended in assay buffer and transferred into FACS tubes. FACS Calibur (Becton&Dickinson) was employed for the measurement.
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