SiR-Actin Kit

SiR-Actin Kit
$0.00
SKU
CY-SC001

SiR-actin is based on the F-actin binding natural product jasplakinolide. SiR-actin is fluorogenic, cell permeable and highly specific for F-actin. Sir-actin stains endogenous F-actin without the need for genetic manipulation or overexpression. Its emission in the far red minimizes phototoxicity and sample autofluorescence. SiR-actin is compatible with GFP and/or m-cherry fluorescent proteins. It can be imaged with standard Cy5 filtersets. SiR-actin can be used for widefield, confocal, SIM or STED imaging in living cells and tissue. Probe quantity allows 50 – 200 staining experiments.*

 

Optical properties

λabs   652 nm

λEm   674 nm
εmax 1.0·105 mol-1·cm-1

*Based on the following conditions: 0.5 – 1 ml staining solution / staining experiments with 0.5 – 1 uM probe concentration. The number of staining experiments can be further increased by reducing volume or probe concentration.

Cytoskeleton, Inc. is the exclusive provider of Spirochrome, Ltd. products in North America.

Citations

"Linklater E. et al. (2021)  Rab40–Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration J Cell Biol (2021) 220 (7): e202008060. https://doi.org/10.1083/jcb.202008060"

Eggers N. et al. (2021)  Cell-free genomics reveal intrinsic, cooperative and competitive determinants of chromatin interactions Nucleic Acids Research, gkab558, https://doi.org/10.1093/nar/gkab558

Tsuchiya K. et al (2021) Ran-GTP Is Non-essential to Activate NuMA for Mitotic Spindle-Pole Focusing but Dynamically Polarizes HURP Near Chromosomes Volume 31, Issue 1, 2021, Pages 115-127.e3, https://doi.org/10.1016/j.cub.2020.09.091

Jin J. et al. (2021) Pulsating fluid flow affects pre-osteoblast behavior and osteogenic differentiation through production of soluble factors https://doi.org/10.14814/phy2.14917

Fläschner, G.et al.  (2021) Rheology of rounded mammalian cells over continuous high-frequencies. Nat Commun 12, 2922 (2021). https://doi.org/10.1038/s41467-021-23158-0

Damenti M. et al. (2021) STED and parallelized RESOLFT optical nanoscopy of the tubular endoplasmic reticulum and its mitochondrial contacts in neuronal cells Neurobiology of Disease,Volume 155,2021,105361,ISSN 0969-9961,https://doi.org/10.1016/j.nbd.2021.105361.

Wurzer H. et al. (2021) Intrinsic Resistance of Chronic Lymphocytic Leukemia Cells to NK Cell-Mediated Lysis Can Be Overcome In Vitro by Pharmacological Inhibition of Cdc42-Induced Actin Cytoskeleton Remodeling Front Immunol. 2021; 12: 619069. doi: 10.3389/fimmu.2021.619069

Kozawa K. et al. (2021) The CD44/COL17A1 pathway promotes the formation of multilayered, transformed epithelia https://doi.org/10.1016/j.cub.2021.04.078

Ordas L. et al. (2021) Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1 Cells 2021, 10(6), 1545; https://doi.org/10.3390/cells10061545

Sala F. et al. (2021) Rapid Prototyping of 3D Biochips for Cell Motility Studies Using Two-Photon Polymerization doi: 10.3389/fbioe.2021.664094

Bain J. et al. (2021) Immune cells fold and damage fungal hyphae PNAS April 13, 2021 118 (15) e2020484118; https://doi.org/10.1073/pnas.2020484118

Girardi F.et al. (2021) TGFβ signaling curbs cell fusion and muscle regeneration. Nat Commun 12, 750 (2021). https://doi.org/10.1038/s41467-020-20289-8

Uhl B. et al. (2021) uPA-PAI-1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils https://doi.org/10.15252/emmm.202013110

Scherer KM. et al. (2021) A fluorescent reporter system enables spatiotemporal analysis of host cell modification during herpes simplex virus-1 replication DOI:https://doi.org/10.1074/jbc.RA120.016571

Choi, Y. W. et al. (2021) Senescent Tumor Cells Build a Cytokine Shield in Colorectal Cancer. Adv. Sci. 2021, 8, 2002497. https://doi.org/10.1002/advs.202002497

Y. Rafati et al. "Effect of microtubule resonant frequencies on neuronal cells", Proc. SPIE 11238, Optical Interactions with Tissue and Cells XXXI, 112381E (25 February 2020); https://doi.org/10.1117/12.2546569

J. Liu, X. Huang, L. Chen, and S. Tan, "Deep learning–enhanced fluorescence microscopy via degeneration decoupling," Opt. Express 28, 14859-14873 (2020).

Zheng, Y‐B, Gong, J‐H, Zhen, Y‐S. Focal adhesion kinase is activated by microtubule‐depolymerizing agents and regulates membrane blebbing in human endothelial cells. J Cell Mol Med. 2020; 24: 72287238. https://doi.org/10.1111/jcmm.15273
Logan, Gregory, and Brooke McCartney. “Comparative analysis of taxol-derived fluorescent probes to assess microtubule networks in a complex live three-dimensional tissue.” Cytoskeleton (Hoboken, N.J.) vol. 77,5-6 (2020): 229-237. doi:10.1002/cm.21599

Tejedo, Milvia Iris Alata et al. “3,3'-thiodipropanol as a versatile refractive index-matching mounting medium for fluorescence microscopy.” Biomedical optics express vol. 10,3 1136-1150. 11 Feb. 2019, doi:10.1364/BOE.10.001136

Leite, Sérgio Carvalho et al. “The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter.” Cell reports vol. 15,3 (2016): 490-498. doi:10.1016/j.celrep.2016.03.047

D’Este, E., Kamin, D., Velte, C. et al. Subcortical cytoskeleton periodicity throughout the nervous system. Sci Rep 6, 22741 (2016). https://doi.org/10.1038/srep22741

“Fluorogenic probes for live-cell imaging of the cytoskeleton”; G. Lukinavičius, L.Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell & K. Johnsson; Nature Methods 11, 731–733, 2014.

 

“STED Nanoscopy Reveals the Ubiquity of Subcortical Cytoskeleton Periodicity in Living Neurons”; E. D’Este, D. Kamin, F. Göttfert, A. El-Hady, S. W. Hell; Cell Reports , Volume 10 , Issue 8 , 1246 – 1251, 2015.

 

“A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins”; G. Lukinavičius, K. Umezawa, N. Olivier, A. Honigmann, G. Yang, T. Plass, V. Mueller, L. Reymond, I. R. Corrêa Jr, Z. Luo, C. Schultz, E. A. Lemke, P. Heppenstall, C. Eggeling, S. Manley & K. Johnsson; Nature Chemistry 5, 132–139, 2013.

 

“Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane”; D. S. Gokhin, R. B. Nowak, J. A. Khoory, A. de la Piedra, I. C. Ghiran and V. M. Fowler; Mol. Biol. Cell; February 25, 2015.

 

“A cleavable cytolysin-neuropeptide Y bioconjugate enables specific drug delivery and demonstrates intracellular mode of action”; V. M. Ahrens, K. B. Kostelnik, R. Rennert, D. Böhme, S. Kalkhof, D. Kosel, L. Weber, M. von Bergen and A. G. Beck-Sickinger; J. Control. Release; 209:170-178, 2015.

 

“Red Si–rhodamine drug conjugates enable imaging in GFP cells”; E. Kim, K. S. Yang, R. J. Giedt and R. Weissleder; Chem. Commun., 50, 4504-4507, 2014.

 

“A marginal band of microtubules transports and organizes mitochondria in retinal bipolar synaptic terminals”; M. Graffe, D. Zenisek, and J. Taraska; J. Gen Physiol. Vol. 146 No.1: 109-117, 2015.

 

 

Application Notes

 

“A Bright Dye for Live-Cell STED Microscopy”; S. Pitsch, I. Köster.

Q1. What is STED microscopy and how does it work?

A1. STED microscopy stands for Stimulated Emission Depletion microscopy.  It is one type of super resolution microscopy which allows the capture of images with a higher resolution than conventional light microscopy which is constrained by diffraction of light.  STED uses 2 laser pulses, one is the excitation pulse which excites the fluorophore, causing it to fluoresce.  The second pulse, referred to as the STED pulse, de-excites the fluorophore via stimulated emission in an area surrounding a central focal spot that is not de-excited and thus continues to fluoresce.  This is accomplished by focusing the STED pulse into a ring shape, a so-called donut, where the center focal spot is devoid of the STED laser pulse, conferring high resolution to the fluorescent area (Fig. 1; see Ref. 1 for more details on STED microscopy).

fig1-faq

Figure 1. STED microscopic image of microtubules labeled with SiR-tubulin in human primary dermal fibroblasts.

Q2. Why is the SiR actin (or tubulin) probe good for STED microscopy?

A2. STED microscopy offers the ability to study cellular details on a nanometermolar scale in vivo.  To take advantage of this super resolution microscopy, one must be able to select with high specificity the area to be examined using fluorescent probes.  In addition, the fluorescent probes must be bright, photostable, exhibit no or little phototoxicity, be excited and emit in the far red spectrum.  In addition, if the probe is to be used for live cell imaging (thus avoiding fixation artifacts that occur when cells are fixed), high cell permeability is necessary.  The SiR actin and tubulin probes fulfill all of these requirements.  In short, the combination of STED and SiR probes allows for unparalleled fluorescent visualization of subcellular actin and tubulin/microtubule structures and their physical characterization in living cells, (see Fig. 2 and Ref. 2). 

Neuron_actin_1
Neuron_actin_closup2_1

Figure 2. STED images of cultured rat hippocampal neurons stained with SiR-actin. Bottom image is a close-up view of part of the top image to clearly visualize actin rings (stripes) with 180 nm periodicity. Courtesy Of Elisa D'Este, MPI Biophysical Chemistry, Göttingen.

Q3. What are the filter sets for these probes?

A3. The SiR actin and tubulin probes are visualized with standard Cy5 filters.  Optimal excitation is 650 nm and emission is 670 nm.  We recommend filters with an excitation of 630 + 20 nm and an emission of 680 + 20 nm (Fig. 3).  

faq-fig3

Figure 3. Excitation (blue) and emission (red) spectra for SiR probes.

Q4. Why do the SiR probes have a low background compared to other fluorophores?

A4. SiR probes are excited by and emit light in the near infrared/far red spectral range, thus avoiding the use of shorter wavelengths such as blue and green light that typically autofluoresce, causing higher background signals.  SiR-coupled probes possess two physical states: 1. a non-fluorescent, closed off-state (spirolactone)  and 2. an open, highly fluorescent on-state (zwitterion).  The binding of the probe to its ligand target favors the highly fluorescent open state while the free unbound probe exists in the closed, non-fluorescent state (Fig. 4).  The fluorescence amplification is 100-fold from the unbound to bound state. This results in a highly sensitive biosensor in which the majority of fluorescence occurs only in the bound state (see Refs. 3 and 4). 

faq-fig4

Figure 4. SiR derivatives exist in equilibrium between the fluorescent zwitterionic (open) form (left structure) and the non-fluorescent spiro (closed) form (right structure).

Q5: Are the SiR probes stable at room temperature?

A5: Yes, the probes are stable at room temperature for a few days.  However, it strongly depends on the probe and the solvent.  Thus, it is recommended to store all of the probes or solutions at –20°C.

 

Q6: Are SiR-actin and SiR-tubulin toxic to cells?

A6: Yes, above a certain threshold both probes show some effect on cell proliferation and altered actin or microtubule dynamics.  However, the probes are orders of magnitude less toxic than their parent drug.  In HeLa cells, neither actin nor microtubule dynamics were altered at concentrations below 100 nM.  At this concentration, SiR probes efficiently label microtubules and F-actin, allowing for the capture of high signal to noise images.

 

Q7: Do the probes work on fixed cells?

A7: SiR-actin probes can be used with PFA-fixed cells.  SiR-actin labels F-actin in PFA-fixed cells as efficiently as phalloidin derivatives.  SiR-tubulin labels microtubules only in ethyleneglycol-bis-succinimidyl-succinate (EGS)-fixed cells.  However, a selective labeling of centrosomal microtubules of PFA-fixed cells was observed.  SiR-actin and SiR-tubulin are not suitable for methanol-fixed cells.

 

Q8: Is it possible to image SiR-probes by STORM?

A8: No—under the very high light intensities typically used in STORM imaging, a phototoxic effect is observed on live cells.

 

Q9: Which organisms and tissues are stained by SiR-probes?

A9: This list describes only cell lines, tissues, or organisms that have been reported to work.  Omission of a cell line, tissue, or organism does not mean that the SiR-probes will not work with the specific cells, tissues, or organisms.

 

Homo sapiens: U2OS, fibroblasts, HeLa, HUVEC, MCF-10A, HCT-116, A549, erythrocytes

Mus musculus: C2C12, IA32, skeletal muscle, primary cardiomyocyte, primary oocyte

Rattus norvegicus: primary hippocampal neurons, primary cortical neurons, NRK

Cercopithecus aethiops: COS-7

Mesocricetus auratus: BHK

Drosophila melanogaster: Notum epithelium, S2

Didelphis marsupialis: OK cells

 

Q10. Do SiR-probes work in 3D cell cultures?

A10: Yes, the probes are able to stain cells in a 3D growth environment.

 

Q11: What are the correction factors CF260 and CF280for the SiR fluorophore?

A11: CF260 = 0.116 and CF280 = 0.147

 

References

1. Hell S.W. and Wichmann J. 1994. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780-782.

2. D’Este E. et al. 2015. STED nanoscopy reveals the ubiquity of subcortical cytoskeleton periodicity in living neurons. Cell Rep. 10, 1246-1251.

3. Lukinavicius G. et al. 2013. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132-139.

4. Lukinavicius G. et al. 2014. Fluorogenic probes for live-cell imaging of the cytoskeleton.Nature Methods. 11, 731-733.