The cytoskeleton is primarily responsible for maintaining cellular shape and organization, and is composed of various protein filaments such as actin, tubulin, and talin. Here we describe cytoskeletal stains that target cytoskeleton structure key components and have been optimized for use in live cells, allowing researchers to follow cytoskeletal dynamics and behavior, or for end-point assays in fixed and permeabilized cells.
Cytoskeleton introduction
The cytoskeleton structure has roles in support, intracellular transport of vesicles and organelles, cell division, motility, scaffolding, and signaling, making it central to both cell health and disease processes. The cytoskeleton structure consists of several components including 3 types of filaments: microfilaments (actin), microtubules (motor proteins: myosin, kinesin, and dynein), and intermediate filaments (learn more about cytoskeleton marker antibodies) [1]. These filaments can be identified with a range of dye conjugates and fluorescent proteins used for fluorescence microscopy.
Selection guide for cytoskeletal stains
Actin is the major cytoskeletal protein of cells. It has two forms: the G-actin monomer which has a globular structure, and F-actin microfilaments that are made up of polymerized G-actin monomers [2]. Actin is involved in many cellular processes, such as muscle contraction, cell motility, cell division (specifically cytokinesis), cell signaling, and maintaining the cell structure. Fluorescently labeled actin is an important tool for investigating the structural dynamics of the cytoskeleton in live and fixed cells.
Learn more about fixed cell actin stains
Learn more about live cell actin stains
| Name | Target | Ex/EM (nm) | Signal-to-noise ratio | Photostability | Live | Fixed | Fixable |
|---|---|---|---|---|---|---|---|
| Fixed cell stains | |||||||
| Alexa Fluor 350 Phalloidin | F-actin | 346/442 |  | | No | Yes | Yes |
| Alexa Fluor Plus 405 Phalloidin | F-actin | 405/450 |  |  | No | Yes | Yes |
| Alexa Fluor 488 Phalloidin | F-actin | 496/519 |  | | No | Yes | Yes |
| Rhodamine Phalloidin | F-actin | 540/565 | | | No | Yes | Yes |
| Alexa Fluor Plus 555 Phalloidin | F-actin | 555/565 | | | No | Yes | Yes |
| Alexa Fluor 568 Phalloidin | F-actin | 578/600 | | | No | Yes | Yes |
| Alexa Fluor 594 Phalloidin | F-actin | 590/617 | | | No | Yes | Yes |
| Alexa Fluor Plus 647 Phalloidin | F-actin | 650/665 | | | No | Yes | Yes |
| Alexa Fluor 680 Phalloidin | F-actin | 679/702 | | | No | Yes | Yes |
| Alexa Fluor 750 Phalloidin | F-actin | 758/784 | | | No | Yes | Yes |
| Live cell stains | |||||||
| CellLight Actin-GFP, BacMam 2.0 | β-actin | 488/520 | | | Yes | No | Yes |
| CellLight Actin-RFP BacMam 2.0 | β-actin | 555/584 | | | Yes | No | Yes |
| Fixed or live cell stains | |||||||
| CellMask Green Actin Tracking Stain | F-actin | 503/512 | | | Yes | Yes | Yes |
| CellMask Orange Actin Tracking Stain | F-actin | 545/570 | | | Yes | Yes | Yes |
| CellMask Deep Red Actin Tracking Stain | F-actin | 652/669 | | | Yes | Yes | Yes |
| CellMask Actin Variety Pack | F-actin | Various | — | — | Yes | Yes | Yes |
Tubulin forms part of the cytoskeleton structure and is involved in various cellular processes, including vesicle and organelle movement, cell division (mitosis and meiosis), and intracellular transport. Tubulin is composed from the polymerization of alpha- and beta-tubulin isotypes which form the microtubule. The differential expression of these isotypes also allows for the study of tubulin function in disease and drug development [3]. Tubulin fluorescent probes are available for imaging the dynamic rearrangements of microtubules in live cells.
Learn more about tubulin stains
| Name | Target | Ex/EM (nm) | Signal-to-noise ratio | Photostability | Live | Fixed | Fixable |
|---|---|---|---|---|---|---|---|
| CellLight Tubulin | |||||||
| CellLight Tubulin-GFP, BacMam 2.0 | β-tubulin | 488/520 | | | Yes | No | Yes |
| CellLight Tubulin-RFP, BacMam 2.0 | β-tubulin | 555/584 | | | Yes | No | Yes |
| Tubulin Tracker | |||||||
| Tubulin Tracker Green | β-tubulin | 494/522 | | | Yes | No | No |
| Tubulin Tracker Deep Red | β-tubulin | 652/669 | | | Yes | No | No |
| Tubulin Tracker Variety Kit | β-tubulin | Various | — | — | Yes | No | No |
Talin is a cytoskeletal protein that is located at cell junctions. It uses actin and integrins to link the cytoskeleton with the extracellular matrix (ECM) [4]. Talin consists of 2 isoforms: Talin-1, which is expressed in most endothelial cells, and Talin-2, which is expressed in cardiac and skeletal muscle, and brain tissue [4]. It is involved in various cellular processes and signaling pathways such as cell communication and structural changes [4]. Talin fluorescent probes are available for imaging in live cells.
Learn more about talin stains
| Name | Target | Ex/EM (nm) | Signal-to-noise ratio | Photostability | Live | Fixed | Fixable |
|---|---|---|---|---|---|---|---|
| CellLight Talin-GFP, BacMam 2.0 | Talin | 488/520 | | | Yes | No | Yes |
ReadyProbes reagents are ready-to-use solutions in convenient dropper bottles, formulated for room-temperature storage. These are designed to stain cells with no calculations, no dilutions, and no pipetting. Simply add 2 drops per milliliter to your cells and image. A choice of either a green- and red-fluorescence are offered for multiplexing or colocalization studies in fixed cells.
Learn more about ReadyProbes reagents
Actin ReadyProbes protocols
| Name | Target | Ex/EM (nm) | Signal-to-noise ratio | Photostability | Live | Fixed | Fixable |
|---|---|---|---|---|---|---|---|
| ActinGreen 488 ReadyProbes | F-actin | 495/518 | | | No | Yes | Yes |
| ActinRed 555 ReadyProbes | F-actin | 555/565 | | | No | Yes | Yes |
HCS CellMask Stains label the entire cell (cytoplasm and nucleus) for a more thorough description of a cell’s anatomy and provide an accurate backdrop against which the features of interest can be evaluated. These stains can be applied to live cells or after fixation and permeabilization.
Learn more about HCS reagents
HCS CellMask stains protocol
| Name | Target | Ex/EM (nm) | Signal-to-noise ratio | Photostability | Live | Fixed | Fixable |
|---|---|---|---|---|---|---|---|
| HCS CellMask Blue Stain | Nucleus, Cytoplasm | 346/442 | | | Yes | Yes | No |
| HCS CellMask Green Stain | Nucleus, Cytoplasm | 496/519 | | | Yes | Yes | No |
| HCS CellMask Orange Stain | Nucleus, Cytoplasm | 555/565 | | | Yes | Yes | No |
| HCS CellMask Red Stain | Nucleus, Cytoplasm | 590/617 | | | Yes | Yes | No |
| HCS CellMask Deep Red Stain | Nucleus, Cytoplasm | 650/665 | | | Yes | Yes | No |
| HCS CellMask Near-IR Stain | Nucleus, Cytoplasm | 777/794 | | | Yes | Yes | No |
Actin is the major cytoskeletal protein of cells. It has two forms: the G-actin monomer which has a globular structure, and F-actin microfilaments that are made up of polymerized G-actin monomers [2]. Actin is involved in many cellular processes, such as muscle contraction, cell motility, cell division (specifically cytokinesis), cell signaling, and maintaining the cell structure. Fluorescently labeled actin is an important tool for investigating the structural dynamics of the cytoskeleton in live and fixed cells.
Learn more about fixed cell actin stains
Learn more about live cell actin stains
| Name | Target | Ex/EM (nm) | Signal-to-noise ratio | Photostability | Live | Fixed | Fixable |
|---|---|---|---|---|---|---|---|
| Fixed cell stains | |||||||
| Alexa Fluor 350 Phalloidin | F-actin | 346/442 | | | No | Yes | Yes |
| Alexa Fluor Plus 405 Phalloidin | F-actin | 405/450 | | | No | Yes | Yes |
| Alexa Fluor 488 Phalloidin | F-actin | 496/519 | | | No | Yes | Yes |
| Rhodamine Phalloidin | F-actin | 540/565 | | | No | Yes | Yes |
| Alexa Fluor Plus 555 Phalloidin | F-actin | 555/565 | | | No | Yes | Yes |
| Alexa Fluor 568 Phalloidin | F-actin | 578/600 | | | No | Yes | Yes |
| Alexa Fluor 594 Phalloidin | F-actin | 590/617 | | | No | Yes | Yes |
| Alexa Fluor Plus 647 Phalloidin | F-actin | 650/665 | | | No | Yes | Yes |
| Alexa Fluor 680 Phalloidin | F-actin | 679/702 | | | No | Yes | Yes |
| Alexa Fluor 750 Phalloidin | F-actin | 758/784 | | | No | Yes | Yes |
| Live cell stains | |||||||
| CellLight Actin-GFP, BacMam 2.0 | β-actin | 488/520 | | | Yes | No | Yes |
| CellLight Actin-RFP BacMam 2.0 | β-actin | 555/584 | | | Yes | No | Yes |
| Fixed or live cell stains | |||||||
| CellMask Green Actin Tracking Stain | F-actin | 503/512 | | | Yes | Yes | Yes |
| CellMask Orange Actin Tracking Stain | F-actin | 545/570 | | | Yes | Yes | Yes |
| CellMask Deep Red Actin Tracking Stain | F-actin | 652/669 | | | Yes | Yes | Yes |
| CellMask Actin Variety Pack | F-actin | Various | — | — | Yes | Yes | Yes |
Tubulin forms part of the cytoskeleton structure and is involved in various cellular processes, including vesicle and organelle movement, cell division (mitosis and meiosis), and intracellular transport. Tubulin is composed from the polymerization of alpha- and beta-tubulin isotypes which form the microtubule. The differential expression of these isotypes also allows for the study of tubulin function in disease and drug development [3]. Tubulin fluorescent probes are available for imaging the dynamic rearrangements of microtubules in live cells.
Learn more about tubulin stains
| Name | Target | Ex/EM (nm) | Signal-to-noise ratio | Photostability | Live | Fixed | Fixable |
|---|---|---|---|---|---|---|---|
| CellLight Tubulin | |||||||
| CellLight Tubulin-GFP, BacMam 2.0 | β-tubulin | 488/520 | | | Yes | No | Yes |
| CellLight Tubulin-RFP, BacMam 2.0 | β-tubulin | 555/584 | | | Yes | No | Yes |
| Tubulin Tracker | |||||||
| Tubulin Tracker Green | β-tubulin | 494/522 | | | Yes | No | No |
| Tubulin Tracker Deep Red | β-tubulin | 652/669 | | | Yes | No | No |
| Tubulin Tracker Variety Kit | β-tubulin | Various | — | — | Yes | No | No |
Talin is a cytoskeletal protein that is located at cell junctions. It uses actin and integrins to link the cytoskeleton with the extracellular matrix (ECM) [4]. Talin consists of 2 isoforms: Talin-1, which is expressed in most endothelial cells, and Talin-2, which is expressed in cardiac and skeletal muscle, and brain tissue [4]. It is involved in various cellular processes and signaling pathways such as cell communication and structural changes [4]. Talin fluorescent probes are available for imaging in live cells.
Learn more about talin stains
| Name | Target | Ex/EM (nm) | Signal-to-noise ratio | Photostability | Live | Fixed | Fixable |
|---|---|---|---|---|---|---|---|
| CellLight Talin-GFP, BacMam 2.0 | Talin | 488/520 | | | Yes | No | Yes |
ReadyProbes reagents are ready-to-use solutions in convenient dropper bottles, formulated for room-temperature storage. These are designed to stain cells with no calculations, no dilutions, and no pipetting. Simply add 2 drops per milliliter to your cells and image. A choice of either a green- and red-fluorescence are offered for multiplexing or colocalization studies in fixed cells.
Learn more about ReadyProbes reagents
Actin ReadyProbes protocols
| Name | Target | Ex/EM (nm) | Signal-to-noise ratio | Photostability | Live | Fixed | Fixable |
|---|---|---|---|---|---|---|---|
| ActinGreen 488 ReadyProbes | F-actin | 495/518 | | | No | Yes | Yes |
| ActinRed 555 ReadyProbes | F-actin | 555/565 | | | No | Yes | Yes |
HCS CellMask Stains label the entire cell (cytoplasm and nucleus) for a more thorough description of a cell’s anatomy and provide an accurate backdrop against which the features of interest can be evaluated. These stains can be applied to live cells or after fixation and permeabilization.
Learn more about HCS reagents
HCS CellMask stains protocol
| Name | Target | Ex/EM (nm) | Signal-to-noise ratio | Photostability | Live | Fixed | Fixable |
|---|---|---|---|---|---|---|---|
| HCS CellMask Blue Stain | Nucleus, Cytoplasm | 346/442 | | | Yes | Yes | No |
| HCS CellMask Green Stain | Nucleus, Cytoplasm | 496/519 | | | Yes | Yes | No |
| HCS CellMask Orange Stain | Nucleus, Cytoplasm | 555/565 | | | Yes | Yes | No |
| HCS CellMask Red Stain | Nucleus, Cytoplasm | 590/617 | | | Yes | Yes | No |
| HCS CellMask Deep Red Stain | Nucleus, Cytoplasm | 650/665 | | | Yes | Yes | No |
| HCS CellMask Near-IR Stain | Nucleus, Cytoplasm | 777/794 | | | Yes | Yes | No |
Fixed cell cytoskeletal stains
Phalloidin is a bicyclic peptide that, when conjugated to fluorescent dyes, can be used to label actin in fixed and permeabilized cells (Figures 1 and 2). Fluorescent conjugates of phalloidin with Alexa Fluor dyes are preferred F-actin stains for most applications because of their bright signals and photostability across the full spectral range. Phalloidin conjugates will bind to large and small actin filaments with a 1:1 stoichiometry between phallotoxin and actin subunits, but they do not bind G-actin monomers and are not suitable for paraffin-embedded sections.
Figure 1. Alexa Fluor 488 phalloidin fluorescence analysis of bovine pulmonary artery endothelial cells. Actin filaments in bovine pulmonary artery endothelial cells were visualized with Alexa Fluor 488 phalloidin). Nuclei were stained with blue-fluorescent DAPI.
Figure 2. HeLa cells labeled with Alexa Fluor Plus 647 Phalloidin. HeLa cells labeled using NucBlue Live ReadyProbes Reagent and Alexa Fluor Plus 647 Phalloidin show multiplexing capability and staining specificity. Cells were mounted in ProLong Glass Antifade Mountant. Images were generated using an EVOS M7000 Imaging System with an Olympus 60X Apochromat Oil objective using DAPI and Cy5 EVOS light cubes.
Live cell cytoskeletal stains
Fluorescent fusion proteins for actin, tubulin, and talin
CellLight fluorescent fusion proteins are designed for cytoskeleton staining of actin, tubulin, or talin in live cells, enabling researchers to identify the cytoskeletal structure and follow cytoskeletal dynamics. CellLight Actin (Figure 3), CellLight Tubulin, and CellLight Talin (Figures 4 and 5) are ready-to-use constructs that express fluorescent fusion proteins targeted to the N-terminus of beta-actin (41.8 kDa), the N-terminus of beta-tubulin (49.8 kDa), or the C-terminus of talin (40 kDa), respectively.
Introducing CellLight fluorescent fusion proteins involves a simple transfection step using the BacMam technology, and they work like cell stains with minimal toxicity or chemical disruption. These cytoskeletal fusion proteins are compatible with other fluorescent probes for multiplex analysis in live cells, or after formaldehyde fixation for colocalization studies.
Learn more about these and other CellLight fluorescent proteins
Figure 3. Expression of CellLight Actin-GFP. U2OS cells labeled using NucBlue Live ReadyProbes Reagent , CellLight Actin-GFP BacMam 2.0, CellLight Mitochondria-RFP BacMam 2.0, and Tubulin Tracker Deep Red show multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Apochromat Oil objective using DAPI, GFP, RFP, and Cy5 EVOS light cubes.
Figure 4. Expression of CellLight Talin-GFP. HeLa cells labeled using NucBlue Live ReadyProbes Reagent, CellLight Talin-GFP BacMam 2.0, and Tubulin Tracker Deep Red show multiplexing capability and staining specificity. Cell in left center shows a mitotic cell with microtubules assembled into a mitotic spindle with visible astral and kinetochore microtubules, as well as cortical actin and condensed DNA. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 100X Super Apochromat Oil objective using DAPI, GFP, and Cy5 EVOS light cubes.
Figure 5. Expression of CellLight Talin-RFP. HeLa cell labeled using CellLight ER-GFP BacMam 2.0, CellLight Talin-RFP BacMam 2.0, and NucBlue Live ReadyProbes Reagent. Z slices were taken at 300 nm steps using an EVOS FL Auto 2 Imaging System equipped with an Olympus 60X Apochromat Oil objective and a GFP, RFP, and DAPI EVOS light cubes to visualize cellular structures in 3D.
Tubulin stains
Tubulin Tracker reagents are endpoint assays used to identify the tubulin cytoskeleton structure and dynamics in live cells (Figures 6 and 7). These cell-permeant reagents provide intense and uniform staining of polymerized tubulin. Tubulin Tracker Green reagent (taxol/paclitaxel conjugate) is an uncharged, non-fluorescent probe whose lipophilic blocking group is cleaved by nonspecific esterase inside the cell. This results in a paclitaxel charged form that is retained inside the cell better than the uncharged form. Tubulin Tracker Deep Red reagent (taxotere/docetaxel conjugate) is a bright and highly photostable probe that specifically stains polymerized tubulin.
These reagents are not generally used for dynamic cell imaging because they interfere with cellular functions, such as the function of tubulin and cell division. Additionally, they are not recommended for fixation studies since staining is lost during cell fixation.
Figure 6. HeLa cells labeled with Tubulin Tracker Green. HeLa cells were labeled with Tubulin Tracker Green using 250 nM and co-stained with NucBlue Live). High resolution images were acquired on a Zeiss LSM710 confocal system using a 60x/1.42 NA oil immersion objective.
Figure 7. HeLa cells labeled with Tubulin Tracker Deep Red. HeLa cells were labeled with Tubulin Tracker Deep Red using 1 µM and co-stained with NucBlue Live). High resolution images were acquired on a Zeiss LSM710 confocal system using a 60x/1.42 NA oil immersion objective.
Live or fixed cell cytoskeletal stains
Actin tracking
CellMask Actin Tracking Stains allow for fluorescent staining of polymerized actin filaments (F-actin) in live (Figures 8 and 9) or fixed cells. They do not label monomer actin (G-actin). CellMask Actin Tracking Stains are cell permeant reagents that provide specific, bright, and uniform staining of the actin cytoskeleton structure.
Following live cell cytoskeleton staining, cells can be fixed or CellMask actin stains can be used in fixed cells. These actin stains can also be used for multiplexing applications in immunofluorescence (IF), immunocytochemistry (ICC), and immunohistochemistry (IHC) protocols. Additionally, CellMask actin tracking stains can be used in 3D cell culture for 3D imaging.
Workflows are as simple as adding 1X staining solution to cells, incubating for 15–60 minutes, and finished by washing and imaging. CellMask Actin Tracking Stains cause no detectable cytotoxicity for up to 24 hours.
Figure 8. Live cell labeling with CellMask Orange Actin Tracking Stain. Hela cells were grown on a 96-well plate and incubated overnight at 37°C with 5% CO2. The cells were stained with CellMask Orange Actin Tracking Stain at 1X concentration and 1 µM LysoTracker Deep Red stain and Hoechst 34580 for 30 minutes at 37°C. The cells were washed three times with HBSS and imaged on an EVOS M7000 imaging system using 40X objective.
Figure 9. Live cell labeling with CellMask Deep Red Actin Tracking Stain. Hela cells were grown on a 96-well plate and incubated overnight at 37°C with 5% CO2. The cells were stained with CellMask Deep Red Actin Tracking Stain at 1X concentration and 500 nM MitoTracker Orange stain and Hoechst 34580 for 30 minutes at 37°C. The cells were washed three times with HBSS and imaged on an EVOS M7000 imaging system using 40X objective.
References:
- Pollard TD and Goldman RD (2018). Cold Spring Harb Perspect Biol. 10(7):a030288. PMID: 29967009.
- Cooper GM (2000). The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Structure and Organization of Actin Filaments.
- Binarová P and Tuszynski J (2019). Cells. 8(10):1294. PMID: 31652491.
- Haining AWM, Lieberthal TJ, and Hernández, A.d.R. (2016). The FASEB Journal. 30:2073-2085. PMID 27252130.
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