Flow cytometry was performed as previously described (10 (link)). In brief, spleens and lymph nodes were pressed through a 70 μm strainer to generate a single-cell suspension, preincubated with 1G12 antibody to block nonspecific Fc binding, stained with directly conjugated antibodies for 15 minutes at 4°C, and washed twice. Antibodies and other reagents used for flow cytometry are listed in Supplemental Tables 2 and 3 . Staining and washes were performed in PBS with 2% fetal bovine serum (FBS). FoxP3 staining was performed with an intracellular staining kit and according to manufacturer instructions (eBioscience, Thermo Fisher Scientific). Apoptosis was measured via annexin V–488 staining (Invitrogen, Thermo Fisher Scientific) for 15 minutes at room temperature in annexin staining buffer (BD Biosciences). APL formation was assessed using a CYTO-ID detection kit (Enzo Life Sciences). In some cases, transplanted animals were injected with BrdU 30 minutes prior to euthanization and samples processed according to manufacturer’s instructions (BioLegend 370706). Flow data were captured on a Fortessa analyzer (BD Biosciences) and evaluated using FlowJo software (version 10.1, Tree Star).
Cyto id detection kit
Manufactured by Enzo Life Sciences
Sourced in Sweden, United Kingdom, United States
The Cyto-ID detection kit is a fluorescent probe-based assay designed to detect and quantify autophagy in cells. The kit utilizes a proprietary cationic amphiphilic dye that selectively labels autophagic vacuoles, allowing for the monitoring of autophagic activity in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Fortessa analyzer, by BD (1 mentions)
Intracellular staining kit, by Thermo Fisher Scientific (1 mentions)
Murine gm csf, by Miltenyi Biotec (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Torin, by Selleck Chemicals (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
8 protocols using cyto id detection kit
1
Flow Cytometry Analysis of Immune Cells
2
Quantifying Autophagy in Macrophages
3
Immunofluorescence Analysis of Cellular Structures
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Immunofluorescence studies in nNOS+ and MOCK cells assessed the cytoskeleton (F-actin labeling with phalloidin-Alexa-594), ER structure (calnexin), proliferation (Ki67), autophagy (CytoID, LC3b-II) and lysosomes (LysoTracker). Cultures were washed in PBS, fixed in 4% PFA in 1xPBS and immunostained or labeled with the respective dyes. Images were captured on an inverted AxioImager Z1 fluorescence microscope (Zeiss, Jena, Germany). FIJI ImageJ was used for counting Ki67 positive proliferating cells after threshold setting and generation of binary images. Nuclei were counter-stained with DAPI. Autophagy was estimated based on the strength and thickness of LC3b-II or CytoID® (CytoID® detection kit Enzo Life Sciences) positive dots. The CytoID® Autophagy Detection measures autophagic vacuoles and monitors autophagic flux in live cells using a cationic amphiphilic tracer dye that rapidly partitions into autophagic vacuoles where it shows bright fluorescence. The dye only weakly stains lysosomes. Quantification of immunofluorescence images was performed with ImageJ, using the particle counter. Images were split into the channels, transformed into binary images by setting the threshold according to the Isodata or Yen algorithm with minor adjustments. Size inclusion/exclusion thresholds and circularity limits were set for analysis of cells or subcellular structures.
4
Quantifying Autophagic Vacuoles in Cells
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Autophagic vacuoles were measured using the Cyto-ID detection kit (Enzo Life Sciences, Solna, Sweden) in cells transfected with control or Par3 siRNAs, and treated with 40 µM chloroquine for the last 16 h of the experiments. Three days after transfection, harvested cells washed and stained for 30 min with Cyto-ID dye, were analyzed using a BD Accuri CG Plus flow cytometer (BD Biosciences) and quantified by FlowJo software version 10.4.2.
5
Autophagy Evaluation by Flow Cytometry
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RPE-1 cells were starved with HBSS for 16 h, or treated with 320 nM RDS 3337 for 72 h in the presence or absence of bafilomycin A1 (Baf A1; 100 nM). At the end of treatment, cells were analyzed by flow cytometry after single staining with a Cyto-ID detection kit (ENZ-51031-K200, Enzo Life Sciences, Exeter, UK). This assay was optimized for the evaluation of autophagy at the cellular level by flow cytometry using a 488 nm-excitable probe that becomes fluorescent in autophagic vesicles (autophagosomes) produced during autophagy. To detect p62/SQSTM1 levels, cells were analyzed by flow cytometry after fixation with 4% paraformaldehyde in PBS and permeabilization with 0.5% Triton X-100 in PBS for 5 min, with anti-p62/SQSTM1 (rabbit, Cell Signaling Technology) primary antibodies followed by anti-rabbit Alexa Fluor 488 (A11008, Invitrogen, Waltham, MA, USA). A representative experiment among 3 is shown. The bar graph reports the mean ± SD obtained in three independent experiments.
6
Autophagy detection in MEFs
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MEFs from WT and Atg4d-deficient mice were incubated in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) as control and upon two different autophagy-inducing conditions – Torin (Selleckchem, at 250 nM) and EBSS (Sigma-Aldrich)- in a 96 well plate. After 4 h of treatment, CYTO-ID® detection kit (Enzo Life Science, ENZ-51031) was used to stain the autophagic vesicles according to the product manual. Briefly: treatment media was removed; cells were washed with buffer and the detection solution then added. After 30 min of incubation at 37 °C, cells were washed twice with buffer and immediately analyzed in a BD Pathway 435 System (BD Bioscience).
7
Autophagy Quantification in SW48 Cells
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SW48 cells were plated at in per well, in glass bottom black-walled 96 well plate (CellVis) and allowed to attach for 24 h. Assay was adapted from CytoID detection kit product manual (Enzo Life Sciences). Briefly, half the wells were treated with chloroquine added 1 hr before other treatments. Then individual wells were treated with 100-nM rapamycin, 250-nM ABT-263, 10-nM TAK-228, TAK-228+ABT-263, or a DMSO control (five wells per condition). Plates were incubated for 24 h at 37°C and 5% . The next day, the assay buffer (5-ml assay buffer + 45-ml DI , supplemented with 5% FBS) and CytoID detection solution ( Cyto-ID green detection reagent, Hoechst 33342 nuclear stain into 10-ml assay buffer) were made as per protocol. Wells were washed with assay buffer, before addition of CytoID Detection solution, plates were incubated at 37°C and 5% for 30 min, then washed with
assay buffer, before a final
assay buffer was added for the read. Plate was read on a fluorescence microplate reader (CytoID: ex: 480 nm, em: 530 nm; Hoechst: ex: 340 nm, and em: 480 nm). CytoID signal was normalized to the Hoechst signal in each well to account for altered cell numbers.
assay buffer, before a final
assay buffer was added for the read. Plate was read on a fluorescence microplate reader (CytoID: ex: 480 nm, em: 530 nm; Hoechst: ex: 340 nm, and em: 480 nm). CytoID signal was normalized to the Hoechst signal in each well to account for altered cell numbers.
8
Autophagy Evaluation in RAW264.7 Cells
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RAW264.7 cells were seeded in 6-well plates at a density of 1 × 106 cells/well, treated with rapamycin (100 nM) for 2 h or 3-MA (5 mM) for 6 h and infected with P. aeruginosa for 4 h. The formation of autophagic vacuoles was detected by CytoID detection kit (Enzo Life Sciences, USA). Briefly, CytoID working solution was prepared by mixing 500 μl of PBS with 1 μl of CytoID and mixing eddies. The cells were suspended with 500 μl CytoID working solution and incubated at room temperature for 30 min. The results were determined by flow cytometry.
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