An In Vitro Fluorescence-Based Assay to Measure Plasma Membrane Resealing Efficiency

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Samenvatting

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.

Protocol

1. Preparation

Cell Plating
Note: Human cervical epithelial cells, HeLa and HeLa expressing Histone 2B-GFP (H2B-GFP), were used in this protocol, but this assay can be adapted to other mammalian cells.
1. Detach adherent cells from a 75 cm² cell culture flask by washing the cells with 2 mL of Trypsin-EDTA 0.25%. Replace the used trypsin with 2 mL of fresh trypsin-EDTA 0.25%.
2. Incubate the cells at 37 ˚C for 5 min until the cells have rounded and detached from the flask.
3. Resuspend the cells in 8 mL of growth medium (DMEM containing 10% heat-inactivated fetal bovine serum,....

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Resultaten

Kinetic assay diagram with 96-well plates for cell analysis, image acquisition, and data analysis steps.
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 ex.......