11–14-day-old primary mouse cortical neurons were loaded for 20 min at 37°C with 1 nM TMRE in Hanks Balanced Salt Solution (HBSS), either without (TMRE) or with [TMRE (FCCP)] 10 μM FCCP in the loading bath. After the 20-min loading, cells remained in the same loading bath, and images at 60× were taken for each condition (100-ms exposure times) using a Chroma HQ535/50x:Q565LP:HQ610/75m filter and dichroic set (Bellows Falls, VT, USA). Two images/condition are presented here. Images are the raw 12-bit grayscale images, converted to 8-bit grayscale for presentation using ImageJ. Immediately above each image, a relative intensity profile in color is presented for the whole image area (Metamorph). Note the specific mitochondrial TMRE loading—in neuronal soma and neurites but excluded from the nucleus as expected—that occurs only when mitochondria are polarized. Also note that contrary to conventional “cartoon” imagery of oval-shaped mitochondria, the elongated, fibrillar mitochondrial shape seen here is typical of many cell types (e.g., References 37 and 38). As demonstrated by the grayscale images and corresponding color intensity profiles, in the depolarized (FCCP) condition, mitochondrial TMRE labeling is completely absent reflecting a complete depolarization of Δψ_m, with only trivial signal arising from a small amount of cytoplasmic vesicular labeling that appears only in the FCCP condition. Note also the considerably higher background fluorescence in the FCCP condition, due to the TMRE remaining in the bath rather than being concentrated by mitochondria.

11–14-day-old primary rat cortical neurons were loaded for 30 min at 37°C with 10 μM Rhod123 in normal growth media as described (45). The Rhod123-containing loading medium was removed, and the cultures placed in CO2-insensitive, pH-stable Leibovitz’s L-15 medium (Invitrogen Life Technologies) and incubated at 37°C in room air for 15 min to allow for the Rhod123 fluorescent signal to reach steady state. Using a Chroma HQ560/55x:Q595LP:HQ645/75m filter and dichroic set, CCD images were captured 1/min for 5 min to establish a baseline. After 5 min, oligomycin or FCCP was added at the times indicated by the arrows, which respectively caused the characteristic quenching (hyperpolarization of Δψ_m) and unquenching (depolarization of Δψ_m) responses in F_wc as expected. Average traces of relative F_wc were taken from n = 4 neurons. This figure and legend is modified from Figure 1C in Reference 45, with permission.

11–14-day-old primary mouse cortical neurons were loaded for 45 min at 37°C with 1 μg/mL (1.5 μM) JC-1 in Hanks Balanced Salt Solution (HBSS), either without (JC-1) or with [JC-1 (FCCP)] 10 μM FCCP in the loading bath. After the 45 min loading, cells remained in the same loading bath, and for each field sequential green (JC-1 monomers) and red (JC-1 aggregates) images at 60× were taken for each condition (100-ms exposure times) using HQ480/40x:Q505LP:HQ535/50m (green) and HQ560/55x:Q595LP:HQ645/75m (red) filters and dichroic sets; one green/red overlay image/condition is presented here. Images are the raw 12-bit images, converted to 8-bit for presentation using ImageJ. When combining green/red images, some adjustments to image stretch and tone were made to each green or red image, for overlay display contrast purposes only, to ensure that all red JC-1 aggregates in both conditions are visible. Note that even after 45 min loading at 1 μg/mL (1.5 μM) (Invitrogen product data sheet suggests 1 μg/mL for 30 min for neurons), in neuronal soma but not neurites, there appear to be concentrated JC-1 monomers (green) in polarized mitochondria (since completely depolarized mitochondria will not accumulate significant cationic JC-1. In the neuronal cell body at lower middle of the left-hand image, note the green fibrillar mitochondria labeling, similar in appearance to the TMRE staining in polarized mitochondria in Figure 1). While it is possible this reflects small “real” differences in Δψ_m between these cellular regions, it also possible that due to different S/V ratios in neurites versus soma and the slow permeance of JC-1, dye concentrations in soma mitochondria have not yet reached aggregation threshold (i.e., they have not yet become red), whereas aggregation threshold is reached sooner in neurites. This example highlights some of the complexities of using JC-1 that must be kept in mind. In the depolarized (FCCP) condition, specific mitochondrial JC-1 labeling appears nearly absent as reflected by a near complete loss of both mitochondrially concentrated monomers (green) and aggregates (red). The little bit of aggregate labeling that does exist in the FCCP condition may reflect a few remaining polarized mitochondria, or more likely, nonspecific vesicular labeling similar to that seen with FCCP+TMRE in Figure 1. Though not seen well here due to contrast adjustments, considerably higher background fluorescence again occurred in the FCCP condition, from JC-1 not concentrated by mitochondria.

11–14-day-old primary mouse cortical neurons were loaded for >20 min at 37°C with 1 nM TMRE in HBSS. After the loading period, cells remained in the same loading bath, and using a Chroma HQ535/50x:Q565LP:HQ610/75m filter and dichroic set, CCD images were captured (1 image/min for 7 min) to establish a baseline; this was followed by direct addition of oligomycin (5 μg/mL) or FCCP (10 μM) at the times indicated by the arrows, which respectively caused the characteristic increase (hyperpolarization of Δψ_m) and decrease (depolarization of Δψ_m) of F_m as expected (red trace). These changes, as expected, were mirrored by an opposing decrease and increase in bath (background) TMRE signal (F_ec) (blue trace) as mitochondria absorbed or released TMRE in response to hyperpolarization and depolarization respectively. Monitoring F_m (or F_wc) and F_ec in parallel represents a simple and convenient approach to help verify expected redistribution behavior of these probes.