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Mechanobiology Lab Extracts New Information, and Great Value, from the Versatile Cytation 5

3-March-18

Fibrosis, a term describing tissue that becomes stiff and scarred, is symptomatic of a number of cardiac, liver and respiratory diseases. A very specific form of fibrosis, known as idiopathic pulmonary fibrosis, is the focus of Dr. Daniel Tschumperlin’s Tissue Repair and Mechanobiology lab at the Mayo Clinic in Rochester, MN. This rare and progressive disease, where lung tissue becomes stiff and scarred without an apparent cause, typically targets an aging population and offers a poor prognosis, as lung function declines and the body cannot receive an adequate oxygen supply.

His lab investigates how cells create and interact with the extra-cellular matrix (ECM), and how “stiffness” of a cell’s ECM changes their biology. Pulmonary fibrosis occurs when cells produce and cross-link extra-cellular matrix to create fibrous tissue, then contract or pull on it to add stiffness to the overall tissue. These actions are triggered when the cell translates mechanical signals to changes in gene expression. Two transcription cofactors, YAP and its homologue TAZ, are localized to the nucleus in a stiff environment, whereas in a soft environment, they are pushed into the cytoplasm; and they are believed to be the driving forces regulating the cell’s change. This type of mechano-signaling is typically studied using mechanically responsive fibroblasts cultured on a plastic substrate that emulates a stiff environment.

When research fellow Dr. Andrew Haak joined the Tschumperlin lab in 2015, he was tasked with developing and miniaturizing assays, and increasing throughput to expedite disease characterization and potential therapy identification. To facilitate these goals, he procured BioTek’s Cytation™ 5 Cell Imaging Multi-Mode Reader in collaboration with other investigators at the Mayo Clinic. “My work revolves around Cytation 5,” he notes. “I can extract so much information so fast, and it’s incredibly easy to know if we’re on to something, or if we should just move on to something else.”

Using Cytation 5, Dr. Haak developed a straightforward and high throughput means of quantifying localization of YAP and TAZ in fibroblasts when plated on the hard, plastic substrate. This provides him a platform to test compounds and generate dose response curves quickly compared to the previous labor-intensive, time-consuming manual assay formats. He also performs traction force experiments, where fibroblasts are plated onto an elastic substrate topped by 200 nm green fluorescent beads then imaged at 10x with Cytation 5. When the cells are removed, the substrate snaps back to its lowest energy state, and another image is taken. The difference calculated between the two time points indicates the amount of force that the cells apply to the substrate. Dr. Haak applies this method to many different cells and stitches the images together before running them through specialized mechano-biology quantification software developed by the lab and freely available to the public. “The strength of this instrument is imaging at 4x and 10x so you can view a huge population of cells; and the great value is then extracting data from the images; even more than you may anticipate.” He notes that other researchers are using Cytation 5 for assays such as tracking the movement and coalescence of lung epithelial, endothelial and fibroblasts into 3D organoids, characterizing senescence, quantifying actin formation and more.

He notes that it’s faster and more efficient to use Cytation 5 to answer research questions compared to debating the issue subjectively. In fact, instead of researchers discussing what appears brighter or localized in a static image, or presenting small representative images of a much larger organ, during lab meetings, he suggests using Cytation 5 to extract empirical data so they can better understand the cellular image and offer supporting statistics.

Dr. Haak truly appreciates the high throughput, but asks, “what good is throughput if you don’t have information?” He continues, “Cytation 5 provides us with high throughput along with the ability to extract quantifiable information, and that combined versatility is incredibly valuable.”

Immunostained fibroblasts imaged at 4x

Immunostained fibroblasts imaged at 4x, showing (A) nuclei (blue); (B) YAP and TAZ (green). (C) overlay of both images, showing strong YAP and TAZ nuclear localization.

 

To learn more about Dr. Daniel Tschumperlin’s Tissue Repair and Mechanobiology lab, visit their web site.


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Thanks to Dr. Andrew Haak at Mayo Clinic for sharing his BioTek experience.



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