SiR-actin

Live cell imaging of Actin, Tubulin & DNA – SLAS conference

30/06/2016 By Ali El Baya, PhD Leave a Comment

In the autumn of 2014, we presented new stains to conduct live cell imaging of Actin and Tubulin, which I covered in my post� 2 new Actin and Tubulin live-cell imaging stains – without transfection!

Fig 1: Live MCF10A cells spheroid stained with SiR-DNA and imaged by confocal microscopy. Courtesy of C. Conrad and K. Jechow (Heidelberg).

SiR-Actin and SiR-Tubulin exhibit a number of benefits which have since then be experienced by a huge number of labs all over the world:

No transfection
No washing steps
No toxic effects if used in the concentration range recommended
Excellent brightness
Far-red excitation & emission
Deep tissue penetration and minimal background
Multiple fluorescent stainings with other dyes possible
Compatible with Superresolution microscopic techniques

Since then, Spirochrome has added another stain with the same benefits: SiR-DNA, a far-red, fluorogenic, cell permeable and highly specific probe for DNA (see Fig 1).

SLAS – High Content Screening Conference

From June 27th to June 29th, the SLAS – High Content Screening Conference took place in Dresden/Germany. The conference focussed on new essays and novel microscopy techniques.

Spirochrome and tebu-bio were selected to present a poster about the features of SiR-stains, which are excellent tools for phenotypic screening and high content screening (HCS) approaches.

If you are interested in learning more about these exciting live cell imaging tools, don’t hesitate to download our joint poster:

Fluorogenic probes for live-cell imaging of intracellular targets

Any questions or comments? Please feel free to contact me with the form below!

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One year on: Live Cell Imaging with SiR-actin, SiR-tubulin & SiR-DNA

29/01/2016 By Ali El Baya, PhD 4 Comments

Fig 1: 3D-SIM microscopy image of labeled Actin stress fibers in human primary dermal fibroblasts.

Launched just over a year ago, � SiR-Actin (Fig 1) and SiR-Tubulin (Fig 2) have been available on the market providing the most convenient tools to stain F-actin and Microtubules in living cells. In the meantime,� Spirochrome have launched a third stain based on the SiR-technology – SiR-DNA (Fig 3) to stain DNA in living cells.
These stains meet the central requirements for live cell imaging tools:

Fig 2: 3D-SIM microscopy image of labeled microtubules in primary rat cortex neuron body.

high selectivity
minimal toxicity
fluorogenic for wash-free imaging
applicable in different cell types and tissues
excited by far-red light
suitable for super-resolution microscopy

[Read more…]

Live Cell Imaging: User results with SiR-Actin & SiR-Tubulin

24/09/2015 By Ali El Baya, PhD Leave a Comment

For one year now, � SiR-Actin and SiR-Tubulin, the second generation of tools to stain actin and tubulin in living cells, have been available on the market – and although it may sound like an overstatement, these stains have changed the world of Live Cell Imaging. [Read more…]

Focus on Actin staining and visualization

07/07/2015 By Ali El Baya, PhD Leave a Comment

Actin serves as one of the major cytoskeleton structures. It is a crucial component involved in a plethora of processes in cell biology: stabilizing the cell shape, cell movements (e.g. cell migration) and intracellular movements and transport mechanisms.

Actin is a 43 kDa protein that is very highly conserved between species. Actin has three main isotypes (α-actin, β-actin and γ-actin), which show >90% amino-acid (aa) homology between isotypes and >98% homology within members of a particular isotypic group. [Read more…]

Staining Actin and Tubulin – from WB to Live Cell Imaging

06/05/2015 By Ali El Baya, PhD Leave a Comment

Fig. 1: Immunofluorescence images of mouse Swiss 3T3 cells stained with anti-actin antibody (027AAN01).

Actin and Tubulin, as the major cytoskeleton structures, are crucial components of a plethora of processes in cell biology. Both are very much involved in stabilizing the cell shape, and especially in cell movements (e.g. cell migration) and intracellular movements and transport mechanisms.

Thus visualizing Actin and Tubulin in fixed or living cells and detecting them in biological samples (e.g. in Western Blots) belongs to basic experimental set-ups in Cell Biology.

A broad range of tools is available for all kind of experimental levels, in this post we’ll take a look at some of these you can use from Western Blot to Live Cell Imaging. [Read more…]

User experience of SiR-Actin and SiR-Tubulin Live Cell Imaging

30/04/2015 By Ali El Baya, PhD 13 Comments

SiR-Actin photo from Stefan Hell's group

STED image of cultured rat hippocampal neurons stained with SiR-actin. Actin rings (stripes) with 180 nm periodicity can be seen. Courtesy of Elisa D’Este, MPI Biophysical Chemistry, Goettingen.

Very recently we launched new Live Cell Imaging tools: SiR-Actin and SiR-Tubulin, produced by Spirochrome.

These stains allow you to stain actin and tubulin in living cells without the need to transfect cells – as I described in my previous posts on these tools:

Today, I invite you to take a look at the brilliant results users of the stains have obtained. Some of them have already been published during the past months. [Read more…]

Live cell imaging tool SiR-actin on JBC frontcover

25/02/2015 By Ali El Baya, PhD 4 Comments

Not long ago, in the summer of 2014,� SiR-actin and� SiR-tubulin to stain actin and tubulin in living cells were launched on the market by Spirochrome (represented across Europe by tebu-bio). Now, SiR-actin has already made its way to the front cover of the most recent issue of the Journal of Biological Chemistry. [Read more…]

Verapamil can enhance live cell staining of Actin & Tubulin with SiR-dyes

12/02/2015 By Ali El Baya, PhD 3 Comments

SiR-actin and SiR-tubulin kits now contain Verapamil

STED image (raw data) : axons of rat primary hippocampal neurons stained with SiR-actin at 16 days in vitro

Recently, we were pleased to launch highly innovative tools to stain actin and tubulin in living cells without the need to transfect cells with vectors coding for GFP- or RFP tagged proteins which bind to filamentous cytoskeletal structures. This makes the� SiR stains produced by Spirochrome the only tools available on the market which allow direct live cell imaging of actin and tubulin. I introduced you to this technology, as well as the benefits of SiR-actin and SiR-tubulin, in a recent post� 2 new Actin and Tubulin live-cell imaging stains – without transfection.

Quite a number of cell types have already been successfully stained with SiR dyes, e.g. HeLa cells, Vero cells, BHK cells and a lot more cell lines, as well as primary cells such as HUVECs cells, dermal fibroblasts, and hippocampal neurons.

However, it turned out that some cell types, especially cell lines, do not sufficiently take up the dye. In these cases, the addition of Verapamil usually increases the uptake efficiency significantly and results in satisfying staining. [Read more…]

Actin staining techniques in fixed and living cells

03/02/2015 By Ali El Baya, PhD Leave a Comment

Actin can be stained in living and fixed cells to determine and follow the structure and function of the cytoskeleton. The actin cytoskeleton is a very dynamic and labile structure in the living cell, but it can be fixed by either cold methanol or paraformaldehyde prior to probing or staining for actin structures.

Actin staining in fixed cells

Fluorescent phalloidin binding to F-Actin, Source: Cytoskeleton Inc.

In fixed cells, actin structures can be visualized by actin antibodies, fluorescent phalloidins, or even electron microscopy.
Antibodies recognize both monomer and polymer (filamentous or F-actin) actin and hence tend to have a high background compared to probes that bind only F-actin. Well designed fluorescent phalloidins only bind to the native quaternary structure of F-actin and therefore have a low background. To create the correct fixation conditions for phalloidin binding, paraformaldehyde must be used as the fixative because it retains the quanternary protein structure which is necessary for high affinity. Methanol destroys the native conformation and hence is not suitable for actin staining with phalloidin.� [Read more…]