CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Dilute 4 μl of Cyto-ID® Green Detection Reagent in 1ml of diluted assay buffer and stain the cells for 30 min at 37°C in the dark.

Protocol tips
Dilute 4 μl of Cyto-ID® Green Detection Reagent in 1ml of diluted assay buffer and stain the cells for 30 min at 37°C in the dark.

Publication protocol

Macrophage infection assay
Freshly grown human THP-1 macrophage-like monocytic cells (in the absence of antibiotics) were adjusted to one million cells/ml, then transferred into 8-well tissue culture plates (2 ml/well) and infected with live Gc FA19, FA19 lptA::spec, or the complemented strain (FA19 lptA C’) at an MOI (multiplicity of infection) of 50 then incubated overnight at 37°C with 5% (v/v) CO2. Uninfected cells in triplicate wells were also incubated simultaneously and were used as a minus infection control. Supernatants from infected or uninfected macrophages were harvested and saved at -20°C for determination of chemokines release and cells were washed with PBS, pelleted (1000 x g for 5 min), and saved at -80°C for western blot analysis. In parallel experiments, cells were washed and stained using the Cyto-ID® autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY) following the manufacturer’s instructions in order to monitor autophagic flux. Briefly, infected and uninfected THP-1 cells (at 1 million cells/ml) were washed twice with 1 ml of diluted assay buffer, then resuspended in 100 μl of Microscopy Dual Detection Reagent Solution (prepared by adding 4 μl of Cyto-ID® Green Detection Reagent and 1 μl of Hoechst 33342 nuclear stain to 1 ml of diluted assay buffer supplemented with 5% [v/v] FBS). Cell pellets were gently resuspended and incubated for 30 min at 37°C in the dark. Cells were then washed twice with diluted assay buffer and resuspended in 100 μl of the same assay buffer. One drop (~40 μl) of stained cells was placed on a clean microscope glass slide, overlaid with a coverslip, and sealed immediately. Autophagic flux in stained cells was monitored within 30 min using fluorescence microscopy (Olympus IX8S1F-3, Olympus Corporation, Japan) at 60x magnification and selecting the standard FITC filter for imaging autophagic flux and the DAPI filter for nuclear staining. Images from at least 10 different fields were collected and analyzed for autophagic flux index, which was calculated by counting the number of green fluorescent puncta in each cell in multiple fields and dividing by the total number of cells in those same fields as determined by counting DAPI-stained nuclei.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Autophagy assay cell type - THP 1 using CYTO-ID® Autophagy detection kit from Enzo Life Sciences.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - THP 1 using CYTO-ID® Autophagy detection kit from Enzo Life Sciences. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.