SiR-tubulin is based on the microtubule binding drug Docetaxel. SiR-tubulin is fluorogenic, cell permeable and highly specific for microtubules. Sir-tubulin stains endogenous microtubules without the need for genetic manipulation or overexpression. Its emission in the far red minimizes phototoxicity and sample autofluorescence. SiR-tubulin is compatible with GFP and/or m-cherry fluorescent proteins. It can be imaged with standard Cy5 filtersets. SiR-tubulin can be used for widefield, confocal, SIM or STED imaging in living cells and tissue. Probe quantity allows 50 – 200 staining experiments.*
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.
|Kemble, Samuel et al.||Analysis of Preplatelets and Their Barbell Platelet Derivatives by Imaging Flow Cytometry||Blood Advances||2022||ISSN 2473--9529|
|Dema, Alessandro et al.||Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules||Current Biology||2022||ISSN 1879-0445|
|Králová, J et al.||Sterolight as imaging tool to study sterol uptake, trafficking and efflux in living cells||Scientific Reports||2022||Article Link|
|Safieddine, Adham et al.||A choreography of centrosomal mRNAs reveals a conserved localization mechanism involving active polysome transport||Nature Communications 2021 12:1||2021||ISSN 2041--1723|
|Singh, Divya et al.||Destabilization of Long Astral Microtubules via Cdk1-Dependent Removal of GTSE1 from Their Plus Ends Facilitates Prometaphase Spindle Orientation||Current Biology||2021||ISSN 1879-0445|
|Vukušić, Kruno et al.||Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation||Developmental Cell||2021||ISSN 1878-1551|
|Schiweck, Juliane et al.||Drebrin controls scar formation and astrocyte reactivity upon traumatic brain injury by regulating membrane trafficking||Nature Communications||2021||ISSN 2041-1723|
|Ordureau, Alban et al.||Temporal proteomics during neurogenesis reveals large-scale proteome and organelle remodeling via selective autophagy||Molecular Cell||2021||ISSN 1097-4164|
|Zhao, Huijie et al.||Fibrogranular materials function as organizers to ensure the fidelity of multiciliary assembly||Nature Communications||2021||ISSN 2041-1723|
|Klemm, Lucas C. et al.||Centriole and Golgi microtubule nucleation are dispensable for the migration of human neutrophil-like cells||Molecular Biology of the Cell||2021||ISSN 1939-4586|
|Quidwai, Tooba et al.||A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia||eLife||2021||ISSN 2050-084X|
|Chinen, Takumi et al.||Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly||Journal of Cell Biology||2021||ISSN 1540-8140|
|Jagrić, Mihaela et al.||Optogenetic control of prc1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces||eLife||2021||ISSN 2050-084X|
|Hoffmann, Patrick C. et al.||Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids||eLife||2021||ISSN 2050-084X|
|Scherer, Katharina M. et al.||A fluorescent reporter system enables spatiotemporal analysis of host cell modification during herpes simplex virus-1 replication||2021||PMID 33380421|
|Buijs, Robin R. et al.||WDR47 protects neuronal microtubule minus ends from katanin-mediated severing||Cell Reports||2021||ISSN 2211-1247|
|Miao, Shumin et al.||DIAPH1 regulates chromosomal instability of cancer cells by controlling microtubule dynamics||European Journal of Cell Biology||2021||ISSN 1618-1298|
|Hao, Kai et al.||Cilia locally synthesize proteins to sustain their ultrastructure and functions||Nature Communications||2021||ISSN 2041-1723|
|Tsuchiya, Kenta et al.||Ran-GTP Is Non-essential to Activate NuMA for Mitotic Spindle-Pole Focusing but Dynamically Polarizes HURP Near Chromosomes||Current Biology||2021||ISSN 1879-0445|
|Kalargyrou, Aikaterini A et al.||Nanotube‐like processes facilitate material transfer between photoreceptors||EMBO reports||2021||ISSN 1469--221X|
|Watson, Joseph L. et al.||High-efficacy subcellular micropatterning of proteins using fibrinogen anchors||Journal of Cell Biology||2021||ISSN 1540-8140|
|Ho, Chi Nguyen Quynh et al.||Simulated Microgravity Inhibits the Proliferation of Chang Liver Cells by Attenuation of the Major Cell Cycle Regulators and Cytoskeletal Proteins||International Journal of Molecular Sciences 2021, Vol. 22, Page 4550||2021||ISSN 1422--0067|
|Gurianov, D. S. et al.||PH domain of BCR provides colocalization of full-length BCR with centrosome together with cortactin to facilitate actin-organizing function||Biopolymers and Cell||2021||ISSN 0233--7657|
|Fiege, Jessica K. et al.||Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium||PLOS Pathogens||2021||ISSN 1553--7374|
|Wolf, Bas de et al.||Chromosomal instability by mutations in the novel minor spliceosome component CENATAC||The EMBO Journal||2021||ISSN 1460--2075|
|Blengini, Cecilia S. et al.||Aurora kinase A is essential for meiosis in mouse oocytes||PLOS Genetics||2021||ISSN 1553--7404|
|Siddiqui, Sana et al.||Epithelial miR-141 regulates IL-13–induced airway mucus production||JCI Insight||2021||ISSN 0021--9738|
|Hégarat, Nadia et al.||Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B||The EMBO Journal||2020||ISSN 0261--4189|
|Park, Yeonkyoung et al.||Nonsense-mediated mRNA decay factor UPF1 promotes aggresome formation||Nature Communications||2020||ISSN 2041-1723|
|Ueki, Hiroshi et al.||Multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon IMPRESS||Nature Protocols||2020||ISSN 1754--2189|
|Afanzar, Oshri et al.||The nucleus serves as the pacemaker for the cell cycle||eLife||2020||ISSN 2050-084X|
|Tabdanov, Erdem D. et al.||Engineering Elastic Nano- and Micro-Patterns and Textures for Directed Cell Motility||STAR Protocols||2020|
|Lv, Qi et al.||RNA-binding protein SORBS2 suppresses clear cell renal cell carcinoma metastasis by enhancing MTUS1 mRNA stability||Cell Death & Disease 2020 11:12||2020||ISSN 2041--4889|
|Stiff, Tom et al.||Prophase-Specific Perinuclear Actin Coordinates Centrosome Separation and Positioning to Ensure Accurate Chromosome Segregation||Cell Reports||2020||ISSN 2211-1247|
|Domart, Florelle et al.||Correlating sted and synchrotron xrf nano-imaging unveils cosegregation of metals and cytoskeleton proteins in dendrites||eLife||2020||ISSN 2050-084X|
|Tona, Yosuke et al.||Live imaging of hair bundle polarity acquisition demonstrates a critical timeline for transcription factor EMX2||eLife||2020||ISSN 2050-084X|
|Drutovic, David et al.||Ran GTP and importin β regulate meiosis I spindle assembly and function in mouse oocytes||The EMBO Journal||2020||ISSN 0261--4189|
|Kesarwani, Shubham et al.||Genetically encoded live-cell sensor for tyrosinated microtubules||The Journal of cell biology||2020||ISSN 1540-8140|
|Cao, Yujie et al.||Microtubule Minus-End Binding Protein CAMSAP2 and Kinesin-14 Motor KIFC3 Control Dendritic Microtubule Organization||Current Biology||2020||ISSN 1879-0445|
|Chinen, Takumi et al.||Nu MA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells||The EMBO Journal||2020||ISSN 0261--4189|
|Wagner, Fabienne et al.||Armadillo repeat-containing protein 1 is a dual localization protein associated with mitochondrial intermembrane space bridging complex||PLoS ONE||2019||ISSN 1932-6203|
|Hu, Junyan et al.||Distinct roles of two myosins in C. Elegans spermatid differentiation||PLoS Biology||2019||ISSN 1545-7885|
|Frontalini, F. et al.||Foraminiferal Ultrastructure: A perspective From Fluorescent and Fluorogenic Probes||Journal of Geophysical Research: Biogeosciences||2019||ISSN 2169-8961|
|Dudka, Damian et al.||Spindle-Length-Dependent HURP Localization Allows Centrosomes to Control Kinetochore-Fiber Plus-End Dynamics||Current Biology||2019||ISSN 0960-9822|
|Khatri, Natasha et al.||The autism protein Ube3A/E6AP remodels neuronal dendritic arborization via caspase-dependent microtubule destabilization||Journal of Neuroscience||2018||ISSN 1529-2401|
|Bennabi, Isma et al.||Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment||EMBO reports||2018||ISSN 1469--221X|
|Drpic, Danica et al.||Chromosome Segregation Is Biased by Kinetochore Size||Current Biology||2018||ISSN 0960-9822|
|Du Toit, André et al.||The precision control of autophagic flux and vesicle dynamics—A micropattern approach||Cells||2018||ISSN 2073-4409|
|Nguyen, Alexandra L. et al.||Genetic Interactions between the Aurora Kinases Reveal New Requirements for AURKB and AURKC during Oocyte Meiosis||Current Biology||2018||ISSN 0960-9822|
|Larsson, Veronica J. et al.||Mitotic spindle assembly and γ-tubulin localisation depend on the integral nuclear membrane protein Samp1||Journal of Cell Science||2018||ISSN 1477-9137|
|Okumura, Masako et al.||Dynein–dynactin–NuMA clusters generate cortical spindle-pulling forces as a multiarm ensemble||eLife||2018||ISSN 2050-084X|
|Hsu, Hsiang Ting et al.||Measurement of lytic granule convergence after formation of an NK cell immunological synapse||Methods in Molecular Biology||2017||ISSN 1064-3745|
|Long, Alexandra F. et al.||Hec1 Tail Phosphorylation Differentially Regulates Mammalian Kinetochore Coupling to Polymerizing and Depolymerizing Microtubules||Current Biology||2017||ISSN 0960-9822|
|Sampson, Josephina et al.||Hsp72 and Nek6 cooperate to cluster amplified centrosomes in cancer cells||Cancer Research||2017||ISSN 1538-7445|
|Elting, Mary Williard et al.||Mapping Load-Bearing in the Mammalian Spindle Reveals Local Kinetochore Fiber Anchorage that Provides Mechanical Isolation and Redundancy||Current Biology||2017||ISSN 0960-9822|
|Kollareddy, Madhu et al.||The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis||Cancer Letters||2017||ISSN 1872-7980|
|Magliocca, Valentina et al.||Identifying the dynamics of actin and tubulin polymerization in iPSCs and in iPSC-derived neurons||Oncotarget||2017||ISSN 1949-2553|
|Stojkov, Darko et al.||ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation||Journal of Cell Biology||2017||ISSN 1540-8140|
|Segal, Dagan et al.||Adhesion and Fusion of Muscle Cells Are Promoted by Filopodia||Developmental Cell||2016||ISSN 1878-1551|
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).
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).
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).
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).
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
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.