Dye toxicity testing

We have tested AB9 for possible toxic effects on T24 cells. In one experiment,cells were kept in a dye-containing medium for 24 hours in a plastic cell culture dish. The same area on the dish was imaged before and after the incubation; Fig. S1 shows that the cells have retained their normal shapes and have proliferated in the presence of the dye.

Fig. S1. Proliferation of T24 cells in a dye-containing medium. The left and right images represent the same area of a dish before (left) and after (right) a 24 hr incubation with 5 mg/ml AB9. The images were collected with an x10/0.3 CPlanFl PhC objective on an Olympus IX70 microscope and recorded with a SPOT InsightTM CCD camera (Diagnostic Instruments, Sterling Heights, MI).The length of the scale bar is 100 m.

Fig. S2 shows fluorescently stained mitochondria in control cells and in cells exposed to AB9 for 24 hours. Mitochondria were stained with 1 MMitotracker Orange (Invitrogen, Eugene, OR) for 30 min and imaged on an Olympus Fluoview 500 confocal microscope using a 543 nm HeNe laserand an x60/1.4 oil-immersion objective. Despite the presence of a dye, the morphology of mitochondria was not affected.

Fig. S2. Mitochondria in T24 cells that had been kept in the dye for 24 hr (left) compared to control (right). The length of the scale bar is 20 m.

All the data presented in the main text have been obtained using 5 mg/ml AB9. Cells also remained healthy after 24 hrs in a medium containing 10 mg/ml AB9 (light staining appears to be due to endocytosis) but not 20 mg/ml AB at which many cells becamepermeable to the dye and developed abnormal features (Fig. S3).

Fig. S3. Images of T24 cells in Hoffman modulation contrast (x20) after exposure to indicated concentrations of AB9 for 24 hours. The length of the scale bar is 50 m.

Survival of T24 cells in the presence of AB9 was quantified using the Trypan Blue exclusionassay [S1], and the results are presented in Fig. S4. Cell viability after a continuous 24-hr exposure to0 - 10 mg/ml AB9 remained 95% or higher; however, increasing the concentration to 20 mg/ml reduced viability to 20%.

Fig. S4. 24-hr cell survival in the presence of AB9. Between 200 and 400 cells were analyzed for each condition.

Comparison of calcein fluorescence and transmission

We attempted to qualitatively compare transmission-through-dye results with the standard calcein fluorescence method [S2]. Fluorescent labeling was achieved by incubating cell with 2 M calcein AM ester (AnaSpec, Fremont, CA)for 30 min. Because volume measurements using the calceinmethod require low depth of field, we used confocal imaging with a high-NA objective (x60/1.4 oil). Calcein fluorescence was excited with an argon 488 nm line, and transmission images were collected using a HeNe 633 nm laser. The medium over T24 cells was initially isotonic, then it was changed to hypotonic, and at the end,the isotonic conditions were restored.Both transmission and fluorescence images showa volume increase in the hypotonic solution and apartial volume recovery after a return to isotonic (there was little RVD response in T24 cells).

Fig. S4. Representative transmission and fluorescence images of T24 cells that were exposed to an isotonic-hypotonic-isotonic sequence. The results for just one cell marked by an arrow are shown: in transmission images,the numbers represent the relative volume (the initial volume taken as 100) and in fluorescence imagesthey indicate theaverage calcein intensity.

Because T24 cells are relatively thick (often more than 10 m), transmission imaging with a high-NA objective produced blurry images; furthermore, the contrast in these images may not reflect thecell thickness correctly (see main text). Therefore, we collected similar imagesusing much thinner rat aortic smooth muscle cells. As with T24 cells, transfer to a hypotonic medium produced an increase in the volume which manifested as an increased intensity in transmission images and a decreased intensity in confocal images (Fig. S5).

Fig. S5. Transmission and confocal fluorescence images ofsmooth muscle cellstransferred from isotonic to hypotonic solution. The arrow points to the cell for which the numbers are shown.

These results demonstrate correlation between transmission and confocal fluorescence.However, presumably due to high flexibility of the large coverslip, we observed frequent defocusing of the fluorescent signal, especially in thin smooth muscle cells, with a loss or redistribution of the intensity. This circumstance (as well as photobleaching of calcein) made collecting long image sequences difficult.

References:

S1. Coder DM (1997) Assessment of cell viability. Curr Prot Cytometry, Unit 9.2.1

S2. Crowe WE, Altamirano J, Huerto L, Alvarez-Leefmans FJ (1995) Volume changes in single N1E-115 neuroblastoma cells measured with a fluorescent probe. Neuroscience 69:283-296