A high-throughput protocol for monitoring starvation-induced autophagy in real time in mouse embryonic fibroblasts

Abstract

Autophagy measurement has been challenging due to the transient nature of autophagy vesicles, in which degradation of cargo occurs. Here, we present a protocol to monitor starvation-induced autophagy using a live high-throughput microscopy system in a fast and automated manner without the need for sample preparation. We provide a detailed protocol describing the generation of turboGFP-LC3B expressing mouse embryonic fibroblasts (MEFs), the measurement of autophagy over time and the analysis of data. For complete details on the use and execution of this protocol, please refer to Nowosad et al. (2020, 2021).

Keywords: Cell Biology; Cell-based Assays; High Throughput Screening; Microscopy.

Graphical abstract

Figure 1

Processing definition for autophagy assay Images represent MEFs expressing turboGFP-LC3B with and without masks using parameters set up in processing definition. The merge channel shows overlap between the green mask (purple) and the phase mask (yellow). For phase channel analysis, the following parameters were applied: segmentation adjustment: 1.1; adjust size: −1 pixel; area: min 200 μm. For green channel analysis, the following parameters were used: Top-Hat segmentation; radius: 90 μm; threshold (GCU): 2; area: min 200 μm. Scale bars are 300 μm.

Figure 2

Quantification of autophagy in turboGFP-LC3B MEFs (A) turboGFP-LC3B MEFs were kept either in full or starvation medium (EBSS supplemented with 10% serum and non-essential amino acids) and fluorescent object confluence was measured every 2 h for 72 h. Fluorescent object confluence results were divided by phase confluence results at the same time point and normalized with the first scan (0 h). (B) Immunoblot of LC3B levels in MEFs starved for the indicated times as in (A). Actin was used as loading control.

Figure 3

TurboGFP-LC3B fluorescence during starvation Examples of images showing MEFs expressing turboGFP-LC3B during a 3-day starvation in EBSS supplemented with 10% serum and non-essential amino acids. Scale bars are 300 μm.

Figure 4

Comparison between turboGFP-LC3B and eGFP-LC3B MEFs were infected with plasmids expressing either turboGFP-LC3B (left) or eGFP-LC3B (right) and imaged using the IncuCyte. Representative images are shown. Scale bars are 300 μm.

Figure 5

Impact of cell clustering on turboGFP-LC3B signal (A) MEFs clusters due to incorrect cell seeding technique. Cell clusters exhibit high turboGFP-LC3B fluorescence levels due to induction of autophagy by contact inhibition. (B) Correctly seeded cells. Scale bars are 300 μm.