Leakage Resistant Indicators
Leakage resistance (LeakRes) versions have a special appendage that increases the intracellular retention – well in excess of an hour.
The first Leakage Resistant Ca2+ indicators, Fura-2 LeakRes (Figure 6.5, formerly Fura-PE3) and Indo-1 LeakRes (Figure 6.6, formerly Indo-PE3) were developed by Minta and Poenie in the early 1990s. Compared to the corresponding non-LeakRes versions, the LeakRes indicators are identical in fluorescence prperties but are retained in the cell for hours. In addition to Fura and Indo, TEFLabs has applied this unique technology to our new indicators Asante Calcium Red and Fluo-2. The LeakRes indicators are available as the K+ salt and the AM forms. (Asante Calcium Green has a very high leakage resistance in its base form.)
TEFLabs plans to offer dextran versions of our calcium indicators in the near future. At this time, in lieu of dextran, we offer leakage resistant versions for injection in cell-impermeable salt forms. In particular, we offer our ultra-resistant Asante Calcium Green.
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Ca2+ sensitive salt form.
6.5 Fura-2 LeakRes (Fura-PE3)
6.6 Indo-1 LeakRes (Indo-PE3)
Figure 6.7 Retention of Fura-2 LeakRes and leakage of Fura-2: 322 T lymphoma cells wer loaded with either Fura-2 or Fura-2 LeakRes and set in calcium buffer. Leakage of Fura-2 or Fura-2 LeakRes into the exterior calcium buffer resulted in increased fluorescence overall. This fluorescence was plotted over time.
Figure 6.8 shows the slower decrease in fluorescence due to leakage of indicator. The top row shows images of BPV cells loaded with Fura-2 LeakRes, whereas the bottom row shows BPV cells loaded with Fura-2. Images were taken at every 20 minutes. Fura-2 cells show loss of significant fluorescence by T = 40 minutes. Fura-2 LeakRes cells retain fluorescence even at T = 100 minutes.
The leakage-resistant Ca2+ indicators has have been used frequently since their introduction in 1994; several literature references are provided as a guide to their use. Table 6.2 shows the properties of TEFLabs’ leakage resistant indicators.
Figure 6.8 methods: BPV cells, adhered to coverslips, were loaded with Fura-2 LeakRes(AM) or Fura-2(AM) as described in Materials and Methods. Cells were mounted in a Sykes-Moore chamber and placed on a water-jacketed holder of a Zeiss IM-35 microscope. The temperature was maintained at 37οC in the sample chamber by a thermostatically controlled circulating water bath. Images were acquired with a Hammamatsu SIT camera and a Photon Technology Image Master illumination and acquisition system. Images of the same microscope field were recorded at 360 nm excitation at 20-min intervals beginning immediately after cells were washed. Camera gain and intensifier voltages were set based on the brightness of cells at the first time point and maintained constant thereafter. Between the acquisition of light was blocked by a shutter. (A-F) The upper series of photographs shows the pattern of fluorescence change for Fura-2 LeakRes loaded BPV cells. The lower series of photographs (G-L) shows the corresponding changes in Fura-2 loaded BPV cells.