The mechanism of resistance to pore-forming toxins is poorly understood. Our protocol enables function and mechanism to pore-forming toxins using Leishmania major, a genetically tractable and physiologically relevant system. The main advantage of the cytotoxicity assay is comparing cell viability of multiple phenotypes at a single-cell level in a medium-throughput assay when challenged with toxins in real time.
Membrane-perturbing agents like amphotericin B are frontline therapies for Leishmania. This assay enables the identification of agents that potentiate amphotericin B and/or new membrane-damaging agents. This assay provides insight into areas such as repair response and signaling pathways related to repair responses.
This assay can be applied to protozoans such as Trypanosoma and Naegleria fowleri. People find it difficult to understand the toxin serial dilution series. So the best advice I can give is to draw a schematic of the serial dilution series before you set up the experiment.
To begin, reserve 0.5 milliliters of processed promastigotes in a separate tube as unstained control. Add two milligrams per milliliter propidium iodide, or PI, to a final concentration of 10 micrograms per milliliter to the remaining promastigotes. Vortex for three seconds.
Add processed promastigotes to each well of a V-bottom, 96-well plate or Marsh tubes, and place the plate or tube rack on the ice at an approximately 45-degrees angle from viewing. Add 100 microliters of assay buffer with propidium iodide to each no-toxin control. Verify that the control was correctly added by visually identifying tubes with a total volume of 200 microliters that appear darker in color.
Add the volume of toxin to the highest dilution. Then, serially dilute the toxin. Pipette up and down at least eight times to ensure mixing.
Starting from the lowest toxin concentration, quickly add 100 microliters of toxin to the correct row, and continue until all the toxin has been added to the cells. Seal the plate with sealing tape. After incubating at 37 degrees Celsius for 30 minutes, package and transport the plate to the flow cytometer.
For data acquisition, start with setting up the flow cytometer and acquisition software according to the manufacturer's instructions and per facility policy. Using the unstained L.major promastigote sample, set the gates for forward and side scatter and the initial fluorescent parameters based on the dyes chosen. Using single-stained controls, set the gates for the viability dye and any fluorescently labeled toxins.
Monitor forward scatter versus time for microclogs, and acquire more than 10, 000 gated events for each sample on the cytometer. For data analysis, gate total single-cell L.major promastigotes by gating on forward and side scatter and time as needed. Use height or area as recommended for the flow cytometer.
Identify and gate dead cells as PI high. Organize the dose-response curve in Excel for analysis. Determine the average percent PI high for each condition between the two technical replicates.
Include toxin concentration and average percent specific lysis, along with experimental details and/or raw percent PI high calculations. Label four more columns as Modeled, Residuals, Parameters, and Parameter Values. Verify that the first columns correspond to the experimental parameters, the toxin concentration, and percent specific lysis.
Initialize the parameters L, k, and c by entering the values in the Parameter Values column. In the Modeled column, create the logistic model. In the Residuals column, calculate the square of the difference between the modeled number and the actual specific lysis.
In the Parameter Values column, next to SUM, sum all the values in the Residuals column. In the Parameter Values column, next to LC50, initialize the equation to calculate the LC50 from the determined values. Open the Solver from the Data tab.
Select the set objective to be the cell containing the sum of the residuals calculated. Set it to Min. Change the variable cells for the parameter values of L, k, and c.
For negative k values, modify equations by factoring out negative one from k to change k to positive. Use the GRG Nonlinear Solving method, and click Solve. Check the curve and that the LC50 is automatically calculated.
Verify the fit by graphically plotting both percent specific lysis and modeling against toxin concentration. The sphingolipid-deficient SPT2 knockout promastigotes were sensitive to SLO in both serum-free M199 and Tyrode's buffer. Wild type and SPT2 add-back promastigotes were resistant to SLO in serum-free M199 but had less than 20%specific lysis in Tyrode's buffer.
The SPT2 knockout promastigotes were approximately eightfold more sensitive to SLO in Tyrode's buffer than in M199. These data demonstrate that toxin sensitivity may vary based on the buffer used. A 120-kilodalton band was observed for phospho-MEK in the L.major promastigotes.
For total MEK, bands at about 120 kilodaltons and 55 kilodaltons were observed, which are consistent with the sizes of MRK1 and LmxMKK, respectively. Phospho-ERK detected similar bands, while the ERK antibody staining was not robust in this assay. The most important thing to remember when attempting this procedure is ensuring the proper handling of the toxin.
Leishmania can be utilized as a genetically tractable model to study plasma membrane repair, providing new insights into membrane repair mechanics during Leishmania bacteria interaction in sandfly midgut.
