How do you choose Spirochrome’s actin probes?

Live cell actin imaging can be demanding and each experiment has its own requirements for many different parameters such as temporal resolution, fluorescence background tolerance, toxicity, bleaching rate and so on.

Spirochrome offer different actin probes – including a test kit to allow you to test all three!

SiR-actin: Spirochrome’s landmark actin probe, a true reference in the field. Good labelling and properties, with some cell types better labelled by SiR than others. Includes Verapamil, a broad-spectrum efflux pump inhibitor, as SiR is sensitive to processing and weak staining by the activity of efflux pumps. The Verapamil can have cellular effects.

SPY650-FastAct: SPY range is the second generation of Spirochrome probes. Available in more colours (SPY555, SPY700) and with better photophysics – superior to SiR in most samples. Verapamil is not included as it isn’t usually needed or desirable due to the superior brightness. The FastAct has better performance with fast actin dynamics.

SPY650-FastAct_X: Spirochrome’s most recent, third generation probe – better performance in long-term cell imaging and sensitive cells. An optimized actin ligand that does not stabilise F-actin and exchanges fast at F-actin binding sites means FastAct_X is the best choice for dynamics and maintaining actin behaviour better than other probes. The lack of F-actin stabilisation means that it is non-cytotoxic and thanks to it’s reduced affinity to actin, exchanges and binds to actin very fast. Therefore, it reduces bleaching kinetics and enhances the speed at which newly polymerized actin is labelled. Only one of the three not suitable for fixation.

Checkout the SPY650-FastAct_X preprint where the probe is described as SiR-XActin here.

Movie of newborn mouse primary cardiac myocytes stained with SiR-actin. High speed (50fps) confocal imaging. Courtesy of Adam Kwiatkowski and Simon Watkins, department of cell biology and center for biologic imaging, University of Pittsburgh.

Timelapse of a CAD cell treated with SPY650-FastAct_X and expressing LifeAct-GFP. Courtesy of Eric Vitriol, Augusta University

Probes Properties

	SiR-actin	SPY650-FastAct	SPY650-FastAct_X
Absorbance maximum λabs	652 nm	652 nm	652 nm
Fluorescence maximum λfl	674 nm	674 nm	674 nm
Works on fixed cells?	yes	yes	no
Fast actin dynamics imaging	+	++	+++
Fluorogenicity	+++	++	+
STED performance with 775 nm depletion	+++	+++	+++
Exchange rate when F-actin bound	slow	fast	fastest
F-actin stabilization	yes	mild	no
Long term imaging	++	++	+++
Shipping	room temperature
Storage	-20°C

Want to know more? Contact sales@kleinaustralia.com.au