AlphaLISA Human and Mouse LC3B-II Detection Kit, 100 assay points

Protocol of the AlphaLISA human and mouse LC3B-II assay

The AlphaLISA Human and Mouse LC3B-II assay can be run in a 96- or 384-well detection plate (50 µL final). As described here, samples (cell lysates or tissue lysates) or control lysate are dispensed directly into the assay plate for the detection of LC3B-II by AlphaLISA reagents. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

Strong correlation between AlphaLISA, HTRF and Western Blot for detection of LC3B-II modulation

U-87 MG cells were plated in a 96-well culture-treated plate (55,000 cells per well) in complete culture medium, and incubated overnight at 37°C, 5% CO₂. The cells were then treated with increasing concentrations of Hydroxycholoroquine (HCQ), a late-stage autophagic inhibitor, for 4 h at 37 °C, 5% CO₂. Following culture medium removal, cells were lysed with 50 µL of 1X Supplemented AlphaLISA Human and Mouse LC3B-II Lysis Buffer for 30 min at room temperature under gentle shaking.

After cell lysis, samples were used for a side-by-side comparison of AlphaLISA LC3B-II, HTRF Human and Mouse LC3B II Detection Kit (#64LC3B2PEG) and Western Blot techniques.

AlphaLISA, HTRF and chemiluminescent signals were normalized with GAPDH Housekeeping protein (HTRF kit: #64GAPDHPEG).

Fold of change corresponds to LC3B-II normalized signal treated over untreated condition.

As evidenced, AlphaLISA LC3B-II, HTRF LC3B-II and Western Blot results are comparable demonstrating that these three methods are correlated.

Induction of autophagy with AZD2014, a mTOR inhibitor

U-87 MG cells were plated in a 96-well culture-treated plate (50,000 cells per well) in complete culture medium, and incubated overnight at 37°C, 5% CO₂. Then, cells were then treated with increasing concentrations of AZD2014 (Vistusertib) for 4 h at 37°C, 5%CO₂. Following culture medium removal, cells were lysed with 50 µl of 1X Supplemented AlphaLISA Human and Mouse LC3B-II Lysis Buffer for 30 min at room temperature under gentle shaking. After cell lysis, samples were then tested with the AlphaLISA LC3B-II.

As expected, AZD2014, a potent mTOR inhibitor, induced autophagy activation, leading to a dose-dependent increase of LC3B-II signal. In addition, no significant cytotoxic effects were observed with ATPlite Luminescence Assay System (#6016736) in the same experimental conditions.

Autophagic flux inhibition with Hydroxychloroquine, a late-stage autophagy inhibitor

U-87 MG cells were plated in a 96-well culture-treated plate (50,000 cells per well) in complete culture medium, and incubated overnight at 37°C, 5% CO₂. Then, cells were then treated with increasing concentrations of Hydroxycholoroquine (HCQ), a late-stage autophagic inhibitor, for 4 h at 37°C, 5%CO₂. Following culture medium removal, cells were lysed with 50 µl of 1X Supplemented AlphaLISA Human and Mouse LC3B-II Lysis Buffer for 30 min at room temperature under gentle shaking. After cell lysis, samples were then tested with the AlphaLISA LC3B-II.

As expected, HCQ, a late-stage autophagic inhibitor, inhibited autophagic flux, leading to a dose-dependent increase of LC3B-II signal. In addition, no significant cytotoxic effects were observed with ATPlite Luminescence Assay System (#6016736) in the same experimental conditions (data not shown).

Autophagy inhibition with ULK-101, a potent and selective ULK1/2 inhibitor

U-87 MG cells were plated in a 96-well culture-treated plate (50,000 cells per well) in complete culture medium, and incubated overnight at 37°C, 5% CO₂. Then, cells then co-treated with 2.5 µM of AZD2014, a mTOR inhibitor, and 10 µM of ULK-101, a potent and selective ULK1/2 inhibitor, for 4 h at 37°C, 5%CO₂. Following culture medium removal, cells were lysed with 50 µl of 1X Supplemented AlphaLISA Human and Mouse LC3B-II Lysis Buffer for 30 min at room temperature under gentle shaking. After cell lysis, samples were then tested with the AlphaLISA LC3B-II.

As expected, ULK-101, a potent and dual autophagy kinase ULK1/2 inhibitor, repressed autophagy induced by AZD2014, leading to a significant decrease of LC3B-II signal. In addition, no significant cytotoxic effects were observed with ATPlite Luminescence Assay System (#6016736) in the same experimental conditions (data not shown).

Specificity of AlphaLISA LC3B-II assay using siRNA

NCI-H1272 cells were plated in a 96-well culture plate (10,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO₂. The day after, the cells were transfected with ON-TARGETplus SMARTPool siRNAs (Horizon Discovery/Revvity) LC3A, LC3B and LC3C isoforms, as well as with a non-targeting siRNA used as negative control using DharmaFECT1 transfection reagent. After a 24h-incubation, the medium was replaced by fresh culture medium, and cells were incubated for an additional 24h-incubation. Then, cells were then treated with 300 µM Hydroxycholoroquine (HCQ), a late-stage autophagic inhibitor, for 4 h at 37°C, 5% CO₂. Following culture medium removal, cells were lysed with 50 µl of 1X Supplemented AlphaLISA Human and Mouse LC3B-II Lysis Buffer for 30 min at room temperature under gentle shaking. After cell lysis, samples were then tested with the AlphaLISA LC3B-II.

The siRNA experiments demonstrate that AlphaLISA detection antibodies specifically measure LC3B isoform and do not recognize the other isoforms. It was found that treatment with the LC3B siRNA induced almost complete AlphaLISA signal loss, while the knockdown of LC3A and LC3C genes did not lead to any significant signal modulation in this assay.

Versatility of AlphaLISA LC3B-II assay using various human and mouse cell lines

U-87 MG (human) and NIH3T3 (mouse) cells were plated in a 96-well culture-treated plate (50,000 cells/well) in complete culture medium, and incubated 48 h at 37°C, 5% CO₂. Then, cells were then treated with 300 µM Hydroxycholoroquine (HCQ), a late-stage autophagic inhibitor, for 4 h at 37°C, 5% CO₂. Following culture medium removal, cells were lysed with 50 µl of 1X Supplemented AlphaLISA Human and Mouse LC3B-II Lysis Buffer for 30 min at room temperature under gentle shaking. After cell lysis, samples were then tested with the AlphaLISA LC3B-II.

The AlphaLISA LC3B-II assay efficiently displayed significant positive signals in each tested cell lines, demonstrating its capability to detect LC3II-B protein in human and mouse and its suitability for translational research.

LC3B-II in autophagy

Autophagy is an evolutionary conserved cellular process that occurs in virtually all eukaryotic cells, ranging from yeast to mammals. Autophagy is crucial in the maintenance of homeostasis, being involved in numerous physiological processes including stress responses (e.g. starvation, hypoxia, high temperature), cell growth, and aging. Conversely, dysfunctions in autophagic mechanisms have been associated with diseases such as cancer, neurodegenerative diseases, infectious diseases, and cardiac and metabolic diseases.

During autophagy, cytoplasmic components, including cytosolic proteins and organelles, are engulfed by autophagosomes. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II). This conjugate is recruited to autophagosomal membranes. Then, autophagosomes fuse with lysosomes to form autolysosomes, and intra-autophagosomal components are degraded by lysosomal hydrolases. At the same time, LC3-II in autolysosomal lumen is degraded. LC3-II level has been found to be proportional to the amount of autophagosomes and thus LC3-II detection has become the most reliable method for monitoring autophagy and autophagy-related processes.