To determine autophagy vacuoles, 2 X 105 hepatocytes were incubated with 1 μl Cyto-ID Green Autophagy detection reagent (Enzo Life Sciences, Exeter, United Kingdom) in 1 ml of HepatoZYME-SFM for 30 min at 37°C, washed, and analyzed by flow cytometry using a Guava EasyCyte Mini System (Millipore, St Charles, MO).
Cyto id green autophagy detection reagent
Manufactured by Enzo Life Sciences
Sourced in United States, United Kingdom
The Cyto-ID Green Autophagy Detection Reagent is a fluorescent dye that specifically labels autophagic vacuoles, enabling the detection and quantification of autophagy in live cells. The reagent provides a sensitive, rapid, and specific method for monitoring autophagic activity.
Automatically generated - may contain errors
Lab products found in correlation
Guava easycyte mini system, by Merck Group (1 mentions)
Flow cytometry, by Beckman Coulter (1 mentions)
Cyto id autophagy detection kit, by Enzo Life Sciences (1 mentions)
Optilyse solution, by Beckman Coulter (1 mentions)
Bz9000 biorevo fluorescence microscope, by Keyence (1 mentions)
Las x, by Leica (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Rapamycin, by Enzo Life Sciences (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Accuri c6, by BD (1 mentions)
7 protocols using cyto id green autophagy detection reagent
1
Quantifying Autophagy Vacuoles in Hepatocytes
2
Autophagy Assessment in Circulating Immune Cells
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Fluorescence of autophagosomotropic dye, Cyto-ID, in circulating immune cells was measured using Cyto-ID™ Autophagy Detection Kit (Enzo Life Sciences, PA, USA) according to the manufacturer’s protocol and the described technique [26 (link), 43 (link)-44 (link)]. Briefly, 100 μL of whole blood was stained with 0.25 μL/mL of Cyto-ID Green Autophagy Detection Reagent (Enzo Life Sciences, PA, USA) and 20 μL of Phycoerythrin-Cyanin 5 (PC5)-conjugated CD45-specific monoclonal antibody (mAb) (Beckman Coulter, Indianapolis IN, USA). After incubation for 30 min in the dark at room temperature, cells were reacted with OptiLyse Solution (Beckman Coulter, Indianapolis IN, USA) for 10 min to lyse red blood cells. After PBS washing, cells were analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA). Monocytes, lymphocytes, and granulocytes were gated by CD45+/side scatters. At least 5 x 104 total cells from each sample were analyzed. The gated lymphocytes, monocytes, and granulocytes were verified as follows: 100 μL samples of whole blood stained with 20 μL of fluorescein isothiocyanate (FITC)-conjugated CD3-specific mAb (Beckman Coulter, USA), 20 μL of PC5-conjugated CD14-specific mAb, and 20 μL of FITC-conjugated CD66b-specific mAb, respectively, with 20 μL of PC5-conjugated CD45-specific mAb separately for 15 min at room temperature. Data were expressed as the MFI of Cyto-ID.
3
Autophagy Quantification by Flow Cytometry
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Up to 1×106 cells were incubated in RPMI (supplemented with 10% FCS and 2 mM L-glutamine but not antibiotics) at 37°C for 30 min in the presence of 0.25 μL/mL Cyto-ID Green Autophagy Detection Reagent (Enzo, UK). Cells were subsequently analysed by flow cytometry. Data are expressed as the mean fluorescence intensity of CytoID divided by the mean forward scatter of the cells, to correct for difference in cell sizes.
4
Autophagy Quantification via Cyto-ID Imaging
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Autophagy activity was detected as described previously [31 ] with some modifications. Cyto-ID Green Autophagy Detection Reagent (Enzo Life Sciences, Farmingdale, NY, USA), a novel amphiphilic autophagosome tracer dye that measures the autophagic vacuoles and monitors autophagic flux in live cells, co-localizes with LC3 and has negligible nonspecific staining of lysosomes [32 (link)], was used according to the manufacturer’s protocol. In brief, small pieces (less than 2 × 2 × 2 mm) of CL tissue samples were incubated in 1× Assay Buffer (Enzo) at 37 C for 30 min in the presence of 2 μl/ml reaction mix. After rinsing in PBS, the stained samples were mounted onto a glass slide and observed immediately under a BZ-9000 Biorevo fluorescence microscope (Keyence) using a 550-nm excitation filter. Images were captured, and fluorescence intensity was analyzed using the ImageJ Software (National Institutes of Health,
Bethesda, MD, USA). For the image analysis, the tissues were selected by setting the threshold above the background level as constant for all analyzed images, and the average fluorescence intensity was calculated to evaluate the autophagy activity.
Bethesda, MD, USA). For the image analysis, the tissues were selected by setting the threshold above the background level as constant for all analyzed images, and the average fluorescence intensity was calculated to evaluate the autophagy activity.
5
Autophagy Assessment in Oocyte Quality
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Autophagy activity in good and poor quality GV oocytes as well as E-64 (Research Institute for the Functional Peptides), rapamycin (Enzo Life Sciences, Farmingdale, NY, USA), E-64 +
rapamycin-treated MII oocytes was examined using Cyto-ID Green Autophagy Detection Reagent (Enzo Life Sciences), an excellent amphiphilic autophagosome tracer dye that
measures the autophagic vacuoles and monitors autophagic flux in live cells [20 (link)], according to the manufacturer’s protocol with some modifications.
Briefly, after removing surrounding cumulus cells, GV and MII oocytes were incubated in 500 μl 1x Assay Buffer (Enzo) including 1 μl/ml reaction mix at 38.5°C in a humidified atmosphere of
5% CO2 and 5% O2 in air for 45 min. Nuclei were stained with 25 μg/ml Hoechst 33342 (Sigma-Aldrich) prepared in reaction medium. After rinsing thrice in phosphate
buffered saline containing 0.2% polyvinyl alcohol (0.2% PVA-PBS), the oocytes were mounted onto a glass slide and observed under a fluorescence microscope using a 550-nm filter (LAS X,
Leica, Germany).
rapamycin-treated MII oocytes was examined using Cyto-ID Green Autophagy Detection Reagent (Enzo Life Sciences), an excellent amphiphilic autophagosome tracer dye that
measures the autophagic vacuoles and monitors autophagic flux in live cells [20 (link)], according to the manufacturer’s protocol with some modifications.
Briefly, after removing surrounding cumulus cells, GV and MII oocytes were incubated in 500 μl 1x Assay Buffer (Enzo) including 1 μl/ml reaction mix at 38.5°C in a humidified atmosphere of
5% CO2 and 5% O2 in air for 45 min. Nuclei were stained with 25 μg/ml Hoechst 33342 (Sigma-Aldrich) prepared in reaction medium. After rinsing thrice in phosphate
buffered saline containing 0.2% polyvinyl alcohol (0.2% PVA-PBS), the oocytes were mounted onto a glass slide and observed under a fluorescence microscope using a 550-nm filter (LAS X,
Leica, Germany).
6
Quantifying Autophagy Induction in Jurkat Cells
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Jurkat T-cells were cultured in RPMI 1640 medium (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS). A total of 1 x 10 6 cells (passage # < 10) was transferred in the wells of a standard 12-well plate. Cells were treated with 10 µM of the autophagy inducer everolimus in the presence or absence of 10 µM test compound. 10 mM of the commonly used autophagy inhibitor 3MA served as a control. After 16 hours of treatment, cells were washed and autophagosomes were stained for 30 minutes with CYTO-ID Green Autophagy Detection Reagent (Enzo Life Sciences, NY, USA) according to the manufacturer's instructions. Finally, cells were washed and analyzed by flow cytometry using an Accuri c6 instrument (BD Biosciences, CA, USA).
7
Autophagy Detection in U251 Cells
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U251 cells were treated with nanoparticles and X-rays as mentioned above. One or three days later, cells were stained with Cyto-ID green autophagy detection reagent (Enzo, Farmingdale, New York), and the autophagy was analyzed with the flow cytometer [10] .
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