Live Imaging Assay for Assessing the Roles of Ca²⁺ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

The overall goal of this procedure is to utilize the lipophilic dye FM 1 43 in live cell imaging to measure the precise kinetics of poor performing toxin removal from the plasma membrane. This is accomplished by first pre binding the toxin at four degrees Celsius. The second step is to transfer the dish to the heated microscope stage and find a focal plane where cells can be visualized.

Next, add the appropriate pre warmed media and immediately begin imaging. The final step is to analyze and quantify intracellular FM 1 43 fluorescence. Ultimately, this sensitive assay can be used to assess the requirements for calcium, fingal, myelin, and other factors for plasma membrane repair.

The main advantage of this technique over existing methods like laser wounding, is that this SLL permeation assay allows synchronized formation of plasma membrane lesions that are very uniform in size after cell ization is triggered. Live time lapse imaging of lipophilic dye influx allows precise determination of kinetics of plasma membrane resealing in a sensitive and very reproducible manner. This assay is ideal for determining the effect of various agents such as calcium and recombinant F mease on plasma membrane repair without the variability associated with other methods of plasma membrane wounding.

To begin the procedure for transcriptional silencing of acid sphingomyelin or a SM seed heela cells in DMEM growth media on 35 millimeter glass bottom dishes incubate overnight at 37 degrees Celsius in a 5%carbon dioxide incubator on the following day, at least one hour before adding the IRNA transfection mixture aspirate off the growth media and replace it with two milliliters of DMEM reduced serum medium. Return the cells to the incubator, prepare four tubes for the SI RNA oligo transfection mixture. The indicated amount of reagent is per 35 millimeter dish to be transfected in tubes A and C, add 250 microliters of optimum reduced serum and four microliters of lipectomy RNAi max in tube B.Add 250 microliters of Optum reduced serum and eight microliters or 160 picomoles of controls in tube D.Add 250 microliters of optimum reduced serum and eight microliters or 160 picomoles of A-S-M-I-A, incubate the tubes at room temperature for five minutes.

Next, combine tubes A and B to form the transfection complex for control S, irna and tube C and D.For the A SMS irna, incubate the reactions at room temperature for 20 minutes. After 20 minutes, add each transfection reaction mixture slowly dropwise into the respective 35 millimeter glass. Bottom dishes incubate at 37 degrees Celsius and 5%carbon dioxide.

At 24 hours post transfection gently aspirate off media and replace with fresh DMEM growth media for efficient A SM knockdown. The dishes should be further incubated at 37 degrees Celsius and 5%carbon dioxide for about 31 hours for a total of 55 hours before live imaging. The following reagents are required for live cell microscopy recombinant PO forming toxin strep lysin O or SLO solution at a concentration of 100 nanograms per microliter in cold PBS without calcium and magnesium.

A 25 millimolar stock solution of FM 1 43 in DMSO solution A, which is FM 1 43, diluted in cold DMEM with calcium to a final concentration of four micromolar and solution B, which is FM 1 43 diluted in cold DMEM without calcium and 10 millimolar EGTA to a final concentration of four micromolar prior to live cell imaging. Prewarm one milliliter aliquots of solution A and solution B in micro centrifuge tubes to 37 degrees Celsius. Keep DMEM with calcium and calcium free DMEM on ice.

Begin preparations for live cell imaging by filling a medium-sized shallow metal container with crushed ice. Invert it into a large glass container with ice and add additional ice, leaving only the bottom surface of the metal container exposed. Place a wet paper towel on the exposed metal bottom to allow for even thermal transfer.

Place one control a treated 35 millimeter glass bottom dish on the cold wet paper towel gently aspirate off all media and wash three times with cold calcium free DMEM after the last wash, aspirate off all media in the dish to image cell associated FM 1 43 in cells with no SLO wounding in the presence of calcium. Add 180 microliters of cold solution B to the center glass cover slip of the 35 millimeter glass bottom dish and incubate for five minutes on ice. Live cell imaging is accomplished with a spinning disc, confocal microscopy system and the appropriate imaging software.

The microscope is equipped with an environmental chamber to maintain temperature, humidity, and carbon dioxide levels. Place the dish on the microscope stage heated to 37 degrees Celsius and replace the lid of the environmental chamber using a 40 x numerical aperture. 1.3 objective.

Find the field of cells that will be imaged For the success of the experiment. An automated focus module on the microscope for correcting thermal drift during imaging is essential If available. Turn on perfect focus or a similar device to correct for thermal drift.

Now remove the cover of the environmental chamber and add one milliliter of prewarm solution a gently to the side of the 35 millimeter dish. Replace the cover of the environmental chamber. Set the parameters for imaging excitation of FM 1 43 is accomplished with a 488 nanometer laser line.

Using a dichroic filter with a 488 nanometer band pass and emission discrimination is achieved through use of a 5 27 emission filter, set laser power levels and camera sensitivity to achieve image exposures of 100 milliseconds. Acquire images for four minutes at one frame every three seconds. The same general protocol is followed for live imaging of cells under different experimental conditions with some modifications as noted in this table to detect FM 1 43 in control S irna treated cells with no SLO wounding in the absence of calcium prewarm, solution B should be added to the side of the 35 millimeter dish.

Instead of solution A prior to imaging to measure FM 43 influx into controls siRNA treated cells wounded with SLO in the presence of calcium, add cold solution B with SLO to the center glass cover slip of the 35 millimeter dish and incubate for five minutes on ice. Then add warm solution A to the dish. Transferring the sample to the warm microscope stage will trigger transmembrane poor formation to measure FM 1 43 influx into control S irna treated cells wounded with SLO In the absence of calcium, add cold solution B with SLO to the center glass cover slip of the 35 millimeter dish and incubate for five minutes on ice.

Then add warm solution B to the dish. To determine the role of a SM in plasma membrane repair, use a SM irna treated cells for all conditions. Lastly, to determine the role of extracellular recombinant bingo myelins on the kinetics of plasma membrane repair, bingo myelin is added to either warm solution A or solution B, and then added to cells.

During live cell imaging in a total volume of one milliliter after movies have been acquired, the intracellular fluorescence intensity of FM 1 43 is measured. Using image analysis software, draw a region of interest in the cytosolic region of each cell in the field and apply the software tools to determine the mean FM 1 43 fluorescence intensity Throughout all frames of the video, select the sample acquisition to be quantified from the list of image sequences on right, double click on the stamp tool to bring up the setup. ROI stamp dialogue box.

For intensity analysis, it is best to select an ellipse stamp. The size of the ellipse must be determined empirically depending upon size and shape of the cell. However, for most tissue culture cells, a four by four micrometer ellipse is appropriate.

It is important to use the same size ROI for each image sequence to be analyzed throughout the quantification. Next, move the time slider to the last time point in order to find the spot of maximal intensity inside the cell with the stamp tool. Highlight an ROI of each cell as pictured.

Now move the time slider back to the zero time point, making sure that the ROI remains within the chosen for the duration of the acquisition. Select measurements. To begin the quantification, a measurements dropdown menu is now available From this dropdown menu, select measure all time points.

Again, click on the measurements dropdown menu and select make measurement item. A dialogue box appears, which allows a name to be given to the data by selecting, okay, a new item containing the data appears with the chosen name. Repeat this sequence for all acquisitions.

For data analysis, select the desired data item from the list on the right, select the analysis tab from the data window to bring up the analysis dropdown menu item. From the analysis dropdown menu, select the analyze menu item and a dialogue box pops up. There are six settings available in the edit analysis box.

First set restrict analysis two to ROIs. For the second box, analyze these data mean should be highlighted. The summarized by box should have value selected.

Finally, the choice of R times should be selected for RO and the choice of name should be selected for column. After these values have been set, click okay to transform the data into a spreadsheet with intensity values per time. For each ROI, the data may also be viewed as a chart By selecting the chart tab in the data window, the data can now be exported as a tab delimited file to be used in a graphical statistical analysis program of the user's choice representative results from the experiments demonstrated are shown here.

Influx of the lipophilic dye FM 1 43 was imaged at one frame per three seconds for four minutes, and FM 1 43 intracellular fluorescence was quantified and expressed as fold increase in fluorescence intensity over time. This first graph shows that in the absence of calcium, both controls IRNA and A SMS irna treated cells were permeable by SLO, which indicates that a SM depletion does not interfere with sensitivity to the toxin. 18 to 27 cells were analyzed in each condition and the error bars correspond to the mean plus or minus SEM.

The results shown in this next graph indicate that in the presence of calcium cells treated with control, S-I-R-N-A were able to reseal their plasma membrane and stop dye influx while cells treated with a SMS irna failed to reseal. Additionally, exogenous addition of low doses of sphingomyelin compliments the plasma membrane defect of a SMS irna treated cells. 18 to 62 cells were analyzed in each condition and the error bars correspond to the mean plus or minus SEM selected timeframes of the movies analyzed are shown in these images.

Interestingly, a full rescue of the ability of a SM deficient cells to reseal after exposure to SLO was observed with low concentrations of Flonase five and 7.5 microunits per milliliter. As the enzyme concentration added to the medium increased, there was a gradual loss in the rescue. Phenotype cells exposed to 10 microunits per milliliter only partially blocked.

The influx of FM 1 43. After exposure to SLO and cells exposed to 50 microunits per milliliter showed a strong dye influx pattern reflecting a full resealing defect similar to that observed in a SM depleted cells. These results suggest that endocytic removal of SLO pores is tightly regulated by the ceramide levels generated at the plasma membrane.

By fcom myelin above a certain level, the process does not occur normally and cells cannot remove the lesions. Remarkably addition of bacterial s Flonase to cells perme by SLO in the absence of calcium also rescued plasma membrane repair. 18 to 31 cells were analyzed in each condition, and the error bars correspond to the mean plus or minus SEM as seen in cells exposed to the pore performing toxin in the presence of calcium only.

Low concentrations of sphingomyelin were effective in blocking influx of FM 1 43. These results indicate that Flonase functions downstream of the calcium dependent step of plasma membrane repair. While attempting this procedure, it is important to have active toxin that was previously tested and titrated to determine the correct working concentration.

This is important because repeated cycles freezing and thawing can inactivate the SLO toxin. After watching this video, you should have a good understanding of how to test plasma membrane, repairability of cells and culture using live sensitive image imaging based assay that allows precise quantification of the kinetics of resealing in a very reproducible manner. You'll also learn how to add specific soluble agents to cells during the imaging procedure and assess their effect on plasma membrane repair.