Optimizing actin probes for live cell fluorescent microscopy
Introduction
The actin cytoskeleton is crucial in various cellular processes, such as cellular migration, phagocytosis, and intracellular trafficking. Aging results in changes in actin expression and dynamics, which can contribute to multiple age-associated diseases and conditions, including cancer, vascular diseases, and neurodegenerative diseases1. Studies in yeast have revealed that the actin cytoskeleton is critical for nutrient metabolism and even lifespan determination2.
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, etc. Choosing the best performing probe for a specific is now simple and cost efficient with Actin_TestKit product. It simply combines our 3 far red actin probes in a single product:
-SiR-actin our landmark actin probe, a true reference in the field
-SPY650-FastAct our 2nd generation actin with better performance with faster actin dynamics.
-SPY650-FastAct_X the last and 3rd generation with an optimized actin ligand that does not stabilize F-actin and exhibits faster exchange. It is highly effective for long term live cell and sensitive cells.
Each actin probe has a different design, specific pros and cons and their overall performance differ greatly from one experiment to the other.
Table 1 below compares their characteristics side by side. It is noted that SPY650-FastAct-X does not stain fixed cell F-actin structures, whereas SiR-Actin and SPY650-FastAct does highlight them, which re-enforces the biochemiscal characteristics of fast dynamics in FastAct-X.
| SIR-Actin | SPY650-FastAct | SPY-650-FastAct_X | |
| Absorbance maximum λabs | 652 nm | 652 nm | 652 nm |
| Fluorescence maximum λfl | 674 nm | 674 nm | 674 nm |
| Imaging "channel" | Cy5 | Cy5 | Cy5 |
| Works on fixed cells? | yes | yes | no |
| Probe quantity | 20 stainings | 20 stainings | 20 stainings |
| Exchange rate when F-actin bound | slow | fast | fastest |
| F-actin stabilization | mild | minimal | none |
| Shipping | room temperature | room temperature | room temperature |
| Storage | -20°C | -20°C | -20°C |
Probe time and concentration optimization
The image below shows how a titration of probe and various time points visualized from T=0 min helps to identify the ideal combination of time and concentration. This image is from Saidjalolov S. et al 2024 and shows a Golgi probe, SiR-AspA (Cat. # CY-SC508) used at 0.1 to 5.0 µM and a time course of 5 to 120 min. A similar approach can be taken for the actin probes but using 1:500 to 1:2000 dilution from the DMSO stock solution, and a timeline of 30 - 360 min.
The optimal combination is a situation where there are still active actin dynamics and cell movement (if thats normal for the cell type), and also bright enough view to allow for multiple time lapse images.
Paper (Saidjalolov S. et al 2024) for titrating probes, see Figure S6, S7 and S8
https://pubs.acs.org/doi/suppl/10.1021/jacsau.4c00487/suppl_file/au4c00487_si_001.pdf
References:
- Lai WF, Wong WT. Roles of the actin cytoskeleton in aging and age-associated diseases. Ageing Res Rev. 2020;58:101021.
- Sing CN, Garcia EJ, Lipkin TG, Huckaba TM, Tsang CA, Coughlin AC, et al. Identification of a modulator of the actin cytoskeleton, mitochondria, nutrient metabolism and lifespan in yeast. Nat Commun. 2022;13(1):2706.
Cells stained with actin (red) and tubulin (green) live cell probes.