High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

This assay was developed to address key questions in the field of plasma membrane biology and to establish how efficiently damaged cells can reseal their plasma membrane. This technique can be used to measure the efficiency of plasma membrane resealing in a high-throughput capacity and to identify specific proteins and corresponding pathways that mediate plasma membrane resealing. Begin by adding 20 milliliters of a 2.5 times 10 to the fifth HeLa cells per milliliter of growth medium suspension into a sterile pipette basin and use a 10 milliliter serological pipette to thoroughly mix the cells.

Next, use a multichannel micropipette to seed 100 microliters of cells per well in triplicate in a 96-well flat, clear bottom, black polystyrene tissue culture treated plate. Remember that a homogenous cell distribution is key to a successful experiment as comparisons between all the experimental conditions require equivalent cell counts. After 24 hours in the humidified cell culture incubator at 37 degrees Celsius and 5%CO2, prewarm the plate reader to 37 degrees Celsius and set the optical configuration to monochromator, the read mode to fluorescence, and the read type to kinetic.