A quantitative TR-FRET plate reader immunoassay for measuring autophagy

Abstract

A utophagy involves the isolation and targeting of unwanted cellular components to lysosomes for their digestion and reuse. Autophagic dysregulation has recently been implicated in a wide range of disease processes, yet facile methods for quantifying autophagy have been lacking in the field. Here we describe the generation of a quantitative plate reader assay for measuring the autophagic activity of cells. One of the best characterized autophagy markers is the protein LC3B, which normally resides in the cytosol (LC3B-I) but upon induction of autophagy becomes lipidated and embedded in autophagosomal membranes (LC3B-II). To quantify autophagy, we reasoned that GFP-tagged LC3B could serve as a time-resolved fluorescence resonance energy transfer (TR-FRET) acceptor upon cell lysis in the presence of terbium-labeled LC3B antibodies. Using this TR-FRET immunoassay approach, we screened a panel of LC3B antibodies and identified an antibody that exhibits strong preferential affinity toward autophagosomeassociated LC3B-II and thereby facilitates specific detection of autophagic activity. The plate reader format provides both a quantitative and an objective result, thus overcoming some of the key limitations of the traditional immunoblotting and imaging approaches used to monitor autophagy. Moreover, since the assay step requires only a single addition of cell lysis buffer containing the detection antibody its simple workflow is both automation-friendly and scalable, which renders it suitable for high-throughput screening. We demonstrate how this TR-FRET immunoassay for GFP-tagged LC3B-II can be applied to quantitatively detect changes in the autophagy activity of cells, including estimating effects on autophagic flux.

Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington's disease models

“…A similar phenomenon was observed in the patient iPSC-derived Q47 neurons ( Figure 6C and 6D ), confirming the effect in a human neuronal model with a polyQ length that is close to the ones commonly found in HD patients. Endogenous LC3 was assessed in these neurons by immunostaining using an antibody that preferentially detects LC3II 52 , because over-expression of LC3-GFP or other LC3 cDNA led to morphological changes and cytotoxicity in these neurons (not shown). Finally, the effect was also confirmed in vivo in heterozygous Hipk3 knockout HD mice ( Figure 6E ).…”

“…The antibody S830 for immunostaining of Htt aggregates is a kind gift from Dr Gillian Bates. Commercially purchased antibodies include anti-HTT antibodies 2166 (Millipore, #MAB2166, RRID:AB_2123255), 3B5H10 (Sigma, #P1874), anti-β-tubulin (Abcam, #ab6046, RRID:AB_2210370), anti-Ataxin3 (Millipore, #MAB5360), anti-Mapk11 (Thermofisher Scientific, #33-8700), anti-Tuj1 (Covance, #MMS-435P, RRID:AB_2313773), anti-Darpp32 (Abcam, #ab40801, RRID:AB_731843), anti-P62 (Abcam, #ab56416, RRID:AB_945626), anti-DAXX (Proteintech, #20489-1-AP, RRID:AB_10693620), anti-LC3B (Proteintech, #18725-1-AP), anti-HIPK3 (Santa cruz, #sc-66920, RRID:AB_2117734) ), anti-β-actin (Abcam, #ab6276), anti-Calnexin (Enzo, #ADI-SPA-860-F), anti-Gapdh (Santa cruz, #sc-25778), anti-α-tubulin (Proteintech, #11224-1-AP), anti-MAP2 (Santa cruz, #sc-20172), anti-phospho-ATF2 (Cell Signaling Technology, #9225, RRID:AB_2060933), anti-ATF2 (Cell Signaling Technology, #9226, RRID:AB_2060930), anti-Phospho-p38 (Cell Signaling Technology, #4511, RRID:AB_2139682), anti-Beclin1 (Thermofisher, #PA1-46464) and anti-LC3 antibody that preferentially detects LC3II (Thermofisher, #70012) 52 . The specificity of all antibodies has been validated by previous reports or our knockdown or knockout experiments.…”

DAMP production by human islets under low oxygen and nutrients in the presence or absence of an immunoisolating-capsule and necrostatin-1

“…Autophagic cells were identified using a Premo TM Autophagy expression kit, time-resolved fluorescence resonance energy transfer of terbium-labeled LC3B antibodies (Tb/GFP TR-FRET LC3B) from Molecular Probes® (Life Technologies TM , NY, USA) 27 . As positive control we applied the lysosomal inhibitor chloroquine (CQ) (half-dilutions from 100-0 μM) to block the turnover of autophagosomes.…”

“…For each well, the TR-FRET emission ratio (518 nm/488 nm) was calculated by dividing the acceptor emission value by the donor emission value. To normalize data, assay window values were determined by dividing the TR-FRET emission ration by the CQ-treated emission ratio values by the average emission ratio of the baseline control 27 .…”

Tools and techniques to measure mitophagy using fluorescence microscopy

“…Using this approach, it is possible to detect either the activation or inhibition of autophagy. 84 Currently there are no means to transfer this technique to study mitophagy, but this is clearly an area that would benefit from further work. Application of the mitochondria-targeted Keima 48 or Rosella 42,64 probes to a plate-reader format may also allow for the high-throughput screening of large compound libraries to identify small-molecule modulators of mitophagy.…”