Determination of SLO Concentrations

The dose-dependence of pore-forming activity for SLO is rather steep. Therefore prepare a series of 1 1 dilutions of SLO in LC. As outlined below, wash the cells with KRH, add 50 xl of your SLO dilutions to the wells for 7 min, aspirate and add trypan blue working solution. Leave the solution for 10 min (for reproducibility, the time of incubation with SLO and with trypan blue is important). Check the degree of permeabilization under the light microscope. The correct dilution of SLO is characterized by 100% of blue cells and minimal loss of cells. 

Remember that precipitates in the trypan blue stock solution will falsify the result, since the final concentration of trypan blue in your working solution will be considerably lower. 

---