Source: Lam, J. G., et al. High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells. J. Vis. Exp. (2019).
This video demonstrates an assay to study the plasma membrane resealing efficiency in mammalian cells. The cells are exposed to a bacterial pore-forming toxin, which forms pores in the cell membrane. In the presence of extracellular calcium ions, the pore causes calcium influx, inducing membrane-resealing events. The resealing efficiency is assessed using DNA-binding propidium iodide dye, which only enters cells without a resealed membrane.
1. Preparation
2. Assay
Note: At the time of the assay, cells must be 70-90% confluent. During the wash steps, the medium should be removed from and applied to the side wall of the well (not directly above the cells). Maintain the temperature of LLO at < 4 ˚C to prevent its aggregation until step 3.1.5.
3. Analysis: Cell Enumeration
Figure 1: Experimental design. The flow diagram depicts a representative plate design configured to test the effect of seven test conditions in comparison to control non-treated cells. Additional controls should be included if appropriate, as for example drug vehicles. Cells are plated (plate 1) 24 h prior to the experiment. On the day of the experiment, cells in plate 1 are washed with M1 or M2 medium pre-warmed at 37 °C, and the plate is imaged (TL, GFP, and PI fluorescence) pre-kinetic. During the 15 min of imaging, reagents are added on ice to plate 2. After imaging, plate 1 is immediately placed on ice for 5 min, and 100 μL/well is transferred from plate 2 to plate 1. Plate 1 is placed in the plate reader to run the kinetic assay at 37 °C for 30 min, followed by imaging (TL, GFP, and PI fluorescence). Data are then analyzed to count cells and assess repair efficiency in all experimental conditions. In large data sets, analysis can be automated. Also, the number of technical replicates can be increased to 4 in high-throughput screens.
The authors have nothing to disclose.
SpectraMax i3x Multi-Mode Microplate Reader | Molecular Devices | i3x | |
MiniMax 300 Imaging cytometer | Molecular Devices | 5024062 | |
TO-PRO-3 | ThermoFisher Scientific | T3605 | |
Propidium Iodide | ThermoFisher Scientific | P3566 | |
HeLa | ATCC | CCL2 | |
HeLa H2B-GFP | Millipore | SCC117 | |
Trypsin-EDTA 0.25% | ThermoFisher Scientific | 25200056 | |
96-well Corning flat bottom black polystyrene tissue culture treated plate | Corning | 3603 | |
Hanks' balanced Salts | Sigma-Aldrich | H4891 | |
EGTA | ISC BioExpress | 0732-100G | |
HEPES | Fisher Scientific | BP310-500 | |
D-(+)-Glucose, HybriMax | Sigma-Aldrich | G5146-1KG |