The near membrane (NearMem) versions have a special lipophilic anchor that tethers the dye to the lipid membrane, so that it can report on changes in [ca2+] ocurring near the membrane. Unfortunately, the high concentrations of calcium influx quickly saturate current near-membrane versions. Design of low affinity near membrane dyes is ongoing.
The first near-membrane fluorescent calcium indicators were bsed on Fura-2 by Minta and Poenie at University of Texas at Austin. The near membrane phenomenom was classically illustrated by Fred Fay, as shown in Figure 6.1, which illustrates the distribution of the dye in the plasma membrane and its response to calcium. Unlike other hydrophobic dyes, TEFLabs near membrane dyes have a piperazine collar to latch the dye into the membrane, leaving the chelating portion to measure calcium in the gates. Our near-membrane UV-excitable indicators Fura-2 NearMem (Figure. 6.9, formerly FFP-18) , and Indo-1 NearMem (Figure. 6.10, formerly FIP-18), have enjoyed considerable success for their applications, because unlike other hydrophobic indicators, they do not get lost in the membrane.
Figure 6.9 Fura-2 NearMem (FFP-18)
Figure 6.10 Indo-1 NearMem (FIP-18)
The applicati12on in neutrophils is a classic example of near-membrane study.
Near membrane versions of Asante Calcium Red, Asante Calcium Green and Fluo-2 are under development. We offer the cell-impermeable potassium salts and the cell-permeable AM form of the near membrane dyes.
Our near-membrane Fura UV indicator Fura-2 NearMem (Figure 6.9), formerly FFP-18 , and Indo-1 NearMem (Figure 6.10) , have enjoyed considerable success for their applications, because unlike other hydrophobic indicators, they do not get lost in the membrane. The piperazine collar prevents such a phenomenon.
Figure 6.11 shows the distribution of the dye Fura-2 NearMem in the plasma membrane. Figure 6.12 is a plot of fluoresence intensity due to Fura-2 NearMem as a function of the distance from the cell membrane. FIG. 6.12 is a plot of the excitation spectra of a titration of Fura-2 NearMem with calcium, where emission was set at 500 nm.
Figure 6.11 Figure 6.12