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

Published: February 29, 2024

Abstract

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. 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%. Incubate the cells at 37 ˚C for 5 min until the cells have …

Representative Results

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 …

Divulgazioni

The authors have nothing to disclose.

Materials

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

Tags

Play Video

Citazione di questo articolo
An In Vitro Fluorescence-Based Assay to Measure Plasma Membrane Resealing Efficiency. J. Vis. Exp. (Pending Publication), e21981, doi: (2024).

View Video