Results in model membranes are consistent with flow cytometry experiments in Escherichia coli and Staphylococcus aureus using a membrane potential sensitive dye (bis-oxonol) and a nucleic acid dye (propidium iodide), suggesting that the mechanism of action is based on membrane binding and collapse of membrane integrity by depolarization and permeabilization.
Fluorescence imaging was performed by staining mitochondria with the membrane potential sensitive dye JC-1 (Invitrogen).
This change in membrane permeability was quantified using a fluorescent, membrane potential sensitive dye, DiBAC4 (3).
To determine changes in Δψm by NEFH down-regulation, intracellular mitochondria were stained with the membrane potential sensitive dye, JC-1, and fluorescence imaging was performed by flow cytometry.
Oocyst infectivity was determined using a cell-culture TaqMan assay (Fig. S4) and Cryptosporidium sporozoites released following oocyst excystation were stained with the membrane potential sensitive dye DiBAC4(3) before flow cytometric analysis to quantify changes to sporozoite membrane polarization.
The cell suspension was incubated with 0.4 µM DiSC3 5) (Dipropylthiacarbocyanine) (Sigma), a membrane potential sensitive cyanine dye, until a stable (approximately 90%) reduction in fluorescence was reached as a result of DiSC3 5) uptake and quenching in the cell due to an intact membrane potential.
A membrane-depolarization assay using a membrane potential-sensitive fluorescent dye revealed that, similar to AmyI-1-18, the antibacterial activity of AmyI-1-18(not) was not dependent on its membrane-disrupting activity.
The former is thought to be released from the endoplasmic reticulum in response to inositol trisphosphate, while the latter enters the cells through a membrane potential-sensitive transporter (Oettgen, H. C., Terhorst, C., Cantley, L. C., and Rosoff, P. M. (1985) Cell 40, 583-590).
Three monoclonal antibodies reactive with different structural domains of the T3-T cell receptor complex of the human T cell leukemia line, HPB-ALL, were previously shown to activate a membrane potential-sensitive, La3+-inhibitable Ca2+ influx (Oettgen, H. C., Terhorst, C., Cantley, L. C., and Rosoff, P. M. (1985) Cell 40, 583-590).
Some MitoTracker dyes, such as MitoTraker®Orange CMTMRos and MitoTraker®Red CMXRos, are mitochondria membrane potential-sensitive, and they lose the staining ability after mitochondria depolarization; While others, such as MitoTraker®Green and MitoTraker®RedFm, accumulate on mitochondria regardless of mitochondrial membrane potential.
To further compare the response to HAMLET between mitochondria and bacteria, depolarization of the mitochondrial membrane potential was quantified as the loss of staining with the membrane potential-sensitive dye TMRE or, using the distribution of tetraphenyl-phosphonium ion.
