Autophagy assay cell type - Macrophages

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

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Autophagy/Cytotoxicity Dual Staining Kit

Cayman Chemicals

Upstream tips	Protocol tips	Downstream tips
	Autophagic vacuoles were detected by incubating cells with MDC solution (1:1,000 in Cell-Based Assay Buffer, 50 μM) using an Autophagy/Cytotoxicity Dual Staining Kit (Cayman Chemical Co.). Cells were incubated with MDC for 10 min at 37˚C, washed three times with PBS and immediately analyzed under a fluorescence Nikon ECLIPSE E600 microscope (Nikon, Tokyo, Japan) equipped with a filter system (excitation wavelength of 365 nm, emission wavelength of 400 nm). For quantification of MDC-positive staining of cells, bright-field and fluorescence images were merged, 100 cells were counted in three separate fields by using a fluorescence microscope and the proportion of MDC-positive cells was determined. Images were captured with a Nikon DIGITAL SIGHT DS-Ri1 microscope camera (Nikon) and imported into Photoshop.

Protocol tips
Autophagic vacuoles were detected by incubating cells with MDC solution (1:1,000 in Cell-Based Assay Buffer, 50 μM) using an Autophagy/Cytotoxicity Dual Staining Kit (Cayman Chemical Co.). Cells were incubated with MDC for 10 min at 37˚C, washed three times with PBS and immediately analyzed under a fluorescence Nikon ECLIPSE E600 microscope (Nikon, Tokyo, Japan) equipped with a filter system (excitation wavelength of 365 nm, emission wavelength of 400 nm). For quantification of MDC-positive staining of cells, bright-field and fluorescence images were merged, 100 cells were counted in three separate fields by using a fluorescence microscope and the proportion of MDC-positive cells was determined. Images were captured with a Nikon DIGITAL SIGHT DS-Ri1 microscope camera (Nikon) and imported into Photoshop.

Protocol tips

Autophagic vacuoles were detected by incubating cells with MDC solution (1:1,000 in Cell-Based Assay Buffer, 50 μM) using an Autophagy/Cytotoxicity Dual Staining Kit (Cayman Chemical Co.). Cells were incubated with MDC for 10 min at 37˚C, washed three times with PBS and immediately analyzed under a fluorescence Nikon ECLIPSE E600 microscope (Nikon, Tokyo, Japan) equipped with a filter system (excitation wavelength of 365 nm, emission wavelength of 400 nm). For quantification of MDC-positive staining of cells, bright-field and fluorescence images were merged, 100 cells were counted in three separate fields by using a fluorescence microscope and the proportion of MDC-positive cells was determined. Images were captured with a Nikon DIGITAL SIGHT DS-Ri1 microscope camera (Nikon) and imported into Photoshop.

Manufacturer protocol Publication protocol 1 Relevant paper

mRFP-green fluorescent protein (GFP)-LC3 adenoviral vector

HanBio Technology

Upstream tips	Protocol tips	Downstream tips
	U937 cells were seeded in 24-well plates and differentiated into macrophages via induction by PMA. To detect autophagic flux, the macrophages were transfected with mRFP-green fluorescent protein (GFP)-LC3 adenoviral vectors according to the manufacturer’s instructions (Hanbio Inc., Shanghai, CN, USA). Successfully transfected macrophages could express LC3 protein tagged by RFP and GFP. GFP is acid sensitive and shows quenching green fluorescence in the acidic environment of a lysosome. However, in contrast with GFP, RFP is relatively stable within lysosomes. Therefore, the number of GFP and RFP puncta was examined and quantified by confocal microscopy. The 'red' and 'yellow' (i.e., a combination of red and green) spots indicated autophagosomes and autolysosomes, respectively.

Protocol tips
U937 cells were seeded in 24-well plates and differentiated into macrophages via induction by PMA. To detect autophagic flux, the macrophages were transfected with mRFP-green fluorescent protein (GFP)-LC3 adenoviral vectors according to the manufacturer’s instructions (Hanbio Inc., Shanghai, CN, USA). Successfully transfected macrophages could express LC3 protein tagged by RFP and GFP. GFP is acid sensitive and shows quenching green fluorescence in the acidic environment of a lysosome. However, in contrast with GFP, RFP is relatively stable within lysosomes. Therefore, the number of GFP and RFP puncta was examined and quantified by confocal microscopy. The 'red' and 'yellow' (i.e., a combination of red and green) spots indicated autophagosomes and autolysosomes, respectively.

Protocol tips

U937 cells were seeded in 24-well plates and differentiated into macrophages via induction by PMA. To detect autophagic flux, the macrophages were transfected with mRFP-green fluorescent protein (GFP)-LC3 adenoviral vectors according to the manufacturer’s instructions (Hanbio Inc., Shanghai, CN, USA). Successfully transfected macrophages could express LC3 protein tagged by RFP and GFP. GFP is acid sensitive and shows quenching green fluorescence in the acidic environment of a lysosome. However, in contrast with GFP, RFP is relatively stable within lysosomes. Therefore, the number of GFP and RFP puncta was examined and quantified by confocal microscopy. The 'red' and 'yellow' (i.e., a combination of red and green) spots indicated autophagosomes and autolysosomes, respectively.

Publication protocol 1 Relevant paper

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream tips
	BMDMs from C57BL/6 J mice were grown in coverslips and treated with compounds with indicated time and autophagy was detected using the cyto-ID Autophagy detection kit (Enzo lifesciences) using FITC channel. Images were taken immediately in Nikon-deconvolution microscope.

Protocol tips
BMDMs from C57BL/6 J mice were grown in coverslips and treated with compounds with indicated time and autophagy was detected using the cyto-ID Autophagy detection kit (Enzo lifesciences) using FITC channel. Images were taken immediately in Nikon-deconvolution microscope.

Manufacturer protocol Publication protocol 1 Relevant paper

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