Novel improved Ca²⁺ indicator dyes on the market-a comparative study of novel Ca²⁺ indicators with fluo-4

Rietdorf, Katja; Chehab, Tala; Allman, Sarah and Bootman, Martin D. (2014). Novel improved Ca²⁺ indicator dyes on the market-a comparative study of novel Ca²⁺ indicators with fluo-4. In: Calcium Signalling: The Next Generation, 9-10 Oct 2014, London, UK.

URL: http://www.biochemistry.org/

Abstract

Recently several new Ca²⁺ indicators were developed, but fluo-3 and fluo-4 still seem to dominate the market. Many of the novel indicators are excited with 488 nm, but there are also indicators excited by longer wavelength (Assante Red, GFP certified FluoForte, Cal-520 Red). Here we present a comparison of some characteristics of these novel indicators with the well-established indicator fluo-4. The new ‘green’ indicators tested were fluo-8, FluoForte, and Cal-520; the ‘red’ indicators were GFP-certified FluoForte, and Cal-520 Red. AM esters of all indicators were loaded into HeLa cells. Cal-520 and FluoForte had a brighter starting fluorescence (153 ± 18% and 146 ± 17%) than fluo-4, whereas fluo-8 is dimmer than fluo-4 (75 ± 9 %). Cal-520 showed the largest F/F₀ increase in response to histamine stimulation, followed by FluoForte, fluo-8 and fluo-4 (6.7 ± 0.3, 5.5 ± 0.7, 4.9 ± 0.6 and 4.7 ± 0.3, respectively). Laser-induced photobleaching was similar for all indicators. GFP-certified FluoForte showed a lower fluorescence when being excited by 488 nm than with 530 nm. However, the F/F₀ response after excitation with both wavelength was similar. We also directly compared four commonly used inhibitors of dye extraction for their efficiency to prevent the loss of fluo-4 at 37 °C. Sulfinpyrazone was the most effective in preventing dye loss (IC₅₀ 144 μM), followed by verapamil (IC₅₀: 240 μM), probenicid (IC₅₀: 353 μM) and sulfobromophtalein (IC₅₀: 2.7 mM).

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