CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry	Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT.	Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.

Upstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Protocol tips
Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT.
Downstream tips
Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.

Publication protocol

5 × 105 CD4+ T-cells were activated in the presence or absence of CQ. Unstimulated cells and cells activated in drug-free medium (positive control) were included as controls. After 24 hours, cells were harvested, washed with PBS + 0.5% BSA + 0.05% NaN3 (all Sigma Aldrich) and incubated with anti-human CD25-PE-Cy7 (clone: BC96), CD69-APC (clone: FN50) and CD71-PE (clone: OKT9, eBioscience).

For measurement of autophagy-associated intracellular vacuoles, we used the Cyto-ID Autophagy Detection Kit (Enzo Life Science, Loerrach, Germany) according to the manufacturers’ instructions. 5 × 105 CD4+ T-cells were activated in the presence or absence of CQ. Unstimulated cells and cells activated in drug-free medium (positive control) were included as controls. At the indicated time points, cells were harvested, washed once in staining buffer and were incubated with a cationic amphiphilic tracer dye for 20 minutes at room temperature. After washing incorporation of the dye into autophagic vacuoles was quantified by flow cytometry.

To determine phosphorylation of intracellular signaling proteins, cells were activated as above (10 μM CQ) and processed as described previously21,23. Cells were harvested and fixated in Fixation Buffer I (BD Phosflow, BD Biosciences) at 37 °C for 10 minutes. After washing in PBS + 0.5% BSA + 0.05% NaN3, cells were re-suspended in pre-chilled (−20 °C) Permeabilization Buffer III (BD Phosflow) and incubated on ice for 30 minutes. Afterwards, cells were washed twice in PBS + 0.5% BSA + 0.05% NaN3 and 20 μL anti-phospho-S6RP (S240; Alexa Fluor 647 conjugated; clone N4-41), anti-phospho-ERK (T202/Y204; Pacific Blue conjugated; clone 20 A) and anti-phospho-p38 (T180/Y182; PE conjugated; clone 36/p38; BD Phosflow) or isotype-matched control antibodies were added for 60 minutes. Subsequently, cells were washed and analyzed on a FACS Canto II flow cytometer.

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Manufacturer protocol

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

Download manufacturer protocol

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