Recently, the non-FRET EnSens technology was launched for in vitro assessment of specific protease activities (Enzium, Inc.). Up to date, the EnSens substates were validated for the detection of protease activity in microplate assays. Now, Enzium has extended the applicability of their Ensens method to 3 D live-cell imaging. On the top of this, they announce that the experimental procedure is easy and non-toxic for cell cultures and co-cultures. So how does live-cell imaging help you locate protease activities in in vitro tumor invasion assays ?
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…]
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:
- 2 new Actin and Tubulin live-cell imaging stains – without transfection!
- Verapamil can enhance live cell staining of Actin & Tubulin with SiR-dyes
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…]
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…]
SiR-actin and SiR-tubulin kits now contain Verapamil
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 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
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…]
Cytoskeletal live-cell imaging is extremely powerful when investigating cellular processes such as cytokinesis, motility and organelle transport and organization. The current experimental procedures remain nevertheless cumbersome and long. This post demonstrates how cell permeable, transfection free, Tubulin and Actin red fluorescent dyes help Cell biologists in analysing cytoskeleton dynamics in living cells.