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 …
Offenlegungen
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