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.
Under the wavelength settings, select a nine nanometer excitation and a 15 nanometer emission bandpass. For assays using propidium iodide, set the excitation and emission wavelengths to 535 nanometers and 617 nanometers, respectively. Under plate type, select 96-wells for the plate format, and select a preset plate configuration corresponding to a black wall clear bottom plate.
Under read area, highlight the wells that will be analyzed throughout the kinetic assay. Under PMT and optics, preset the flashes per read to six and check the box for read from bottom. Under timing, enter zero hours 30 minutes and zero seconds into the total run time for a 30 minute kinetic assay and enter zero hours five minutes and zero seconds for the interval.
Confirm the specified settings in the settings information and click OK.Then click read to initiate the kinetic run. To set the imaging parameters within the settings mode, set MiniMax for the optical configuration, imaging for the read mode, and endpoint for the read type. Under wavelengths, select transmitted light and the appropriate fluorescence boxes corresponding to the experimental excitation and emission wavelengths.
Set the plate type and the read area as just demonstrated and under the well area setting, set the number of sites within a well to be imaged. Under the image acquisition settings for GFP, set the device to image the entire well with an exposure time of 20 milliseconds per image. For transmitted light, acquire a single image of the center of each well with an eight millisecond exposure time.
For propidium iodide, acquire a single image at the center of each well with a 20 millisecond exposure time. Then confirm the settings in the settings information and click OK and read to initiate the imaging. To setup a high-throughput fluorescence-based assay for the repair permissive conditions, wash the cells two times with 200 microliters of 37 degree Celsius M1 medium per well and add 100 microliters of fresh 37 degree Celsius M1 medium supplemented with 30 micromolar propidium iodide after discarding the second wash.
For repair restrictive conditions, wash the cells one time with 200 microliters of 37 degree Celsius M2 medium supplemented with five millimolar EGTA per well followed by one wash with 200 microliters of M2 medium alone. After discarding the second wash, add 100 microliters of fresh 37 degree Celsius M2 medium supplemented with 30 micromolar propidium iodide per well. Then image the plate under transmitted light, GFP, and PI as just demonstrated.
While the plate is being imaged, place a 96 well round bottom polypropylene microplate on ice and configure the plate as just demonstrated for the previous plate. For repair permissive conditions, add 100 microliters of ice cold M1 medium supplemented with 60 micromolar propidium iodide per well followed by the addition of 100 microliters of ice cold M1 with or without 4X listeriolysin O per well. For repair restrictive conditions, add 100 microliters of ice cold M2 medium supplemented with 60 micromolar propidium iodide per well followed by the addition of 100 microliters of ice cold M2 with or without 4X listeriolysin O per well.
It is imperative to prepare listeriolysin O at the appropriate experimental concentration on ice approximately five minutes prior to the start of the kinetic assay when plate one is on ice and plate two is being prepared. When the first plate is finished imaging, immediately transfer the plate onto a sheet of aluminum foil on ice. After five minutes, transfer 100 microliters from each well of the second plate to the appropriate corresponding well in the first cooled plate, inserting the pipette tips below the meniscus of solution in each well before ejecting the volume without mixing for proper distribution of the toxin.
Allow the toxin to bind to the host cells for one minute and immediately transfer the first plate to the plate reader for the post-kinetic assay using the spectrofluorometer mode. At the end of the kinetic assay, immediately acquire a post-kinetic imaging of the plate image as demonstrated for the pre-kinetic imaging. To determine the cell count based on the nuclear fluorescence, in the microplate cell enumeration software under settings, select re-analysis, and in the image analysis settings, select discrete object analysis using 541 as a wavelength for finding objects.
Within the find objects option, use the draw on images finding method, select nuclei, and click apply, then click OK and read to initiate the cell counting algorithm. GFP expression does not interfere with propidium iodide intensity measurements in the presence or absence of listeriolysin O treatment. The expression of propidium iodide can bleed through GFP fluorescence as evidenced by an increase in GFP fluorescence intensity in HeLA H2B-GFP cells in the post-kinetic imaging assay compared to the pre-kinetic analysis.
Importantly however, this crossover does not affect cell counting, as the segmentation process involved in the enumeration of the nuclei is unaffected by an increase in GFP fluorescence. In the absence of listeriolysin O, the plasma membrane integrity remains constant in the presence or absence of extracellular calcium, while listeriolysin O treatment in the presence of calcium supplemented medium results in a steady increase in propidium iodide fluorescence intensity. In the absence of extracellular calcium however, there is a significantly steeper increase in propidium iodide fluorescence, reflecting the absence of membrane resealing.
Alternatively, a carbocyanine nucleic acid binding dye with fluorescence in the far red can be substituted for propidium iodide to minimize the spectral overlap with GFP. In addition, the larger excitation coefficient for the far red dye exhibits a higher signal to noise ratio and a better resolution between the repair permissive and repair restrictive conditions. While attempting this procedure, it is recommended to label all of the tubes a day ahead of experiment, as this will prepare you for all of the reagent preparation on the day of the experiment.
Following this procedure, other methods like image cytometry can be performed to answer additional questions, such as does a pore forming toxin damage all cells uniformly or are some cells more susceptible than others. After its development, this technique has paved the way for researchers in the fields of infectious disease and plasma membrane repair to explore the effects of pore size on the repair efficiency of different cell types.
