Experimental Examples

Restructuring of the Human Macrophage Cytoskeleton During Borreliae Uptake

Borrelia bacteria are the cause of the Lyme disease, also known as Lyme borreliosis. To prevent the dissemination of borreliae, their uptake and elimination by macrophages has been shown to be necessary. This process involves dynamic restructuring of the macrophage cytoskeleton, particularly of the actin microfilaments.

In this experiment, LifeAct was used to visualize actin cytoskeleton reorganization in human macrophages during phagocytosis of borreliae.

Phagocytosis of borreliae by a primary human macrophage. Time-lapse movie of a confocal z-stack showing a primary human macrophage expressing RFP-LifeAct (red) internalizing several GFP-expressing spirochetes (green) with actin-rich cell protrusions. Sequence 41 min. Data by Dr. Mirko Himmel and Prof. Stefan Linder, PhD, Universitätsklinikum Hamburg-Eppendorf, Germany, http://www.linderlab.de/.

Live Imaging of Gap Closure in a Wound Healing Assay

The wound healing and migration assay is a widely-used tool for studying cell migration. In the in vitro analysis of wound closure in an MCF7 cell layer, a gap was created by using the Culture-Insert 2 Well in a µ-Dish ^{35 mm, high}. The ibidi Stage Top Incubator was applied to create and maintain physiologic conditions. To create a time-lapse video of the wound closure, a phase contrast microscopy picture was taken every hour for a total of 29 hours.

Live cell imaging using the ibidi Stage Top Incubator shows the gap closure of MCF7 cells in a wound healing and migration assay. Phase contrast; 10x objective lens.

Live Microscopy of Endothelial Cell Tube Formation

The tube formation assay is a quick and easy-to-perform experiment for analyzing angiogenesis in vitro. In the following example using the µ-Slide 15 Well 3D, endothelial cells (HUVEC) were seeded on a Matrigel^® layer. Physiologic conditions were established and maintained with the ibidi Stage Top Incubator. To create a time-lapse video of the tube formation, a low-resolution phase contrast microscopy picture was taken every 10 minutes for a total of 24 hours.

Live cell imaging using the ibidi Stage Top Incubator shows the tube formation of HUVECs in an angiogenesis assay. Phase contrast; 4x objective lens.

Live Visualization of Cell Migration During a Chemotaxis Assay

A chemotaxis assay is conducted to analyze whether or not a cell type directly orients and migrates towards a defined chemoattractant. Here, the µ-Slide Chemotaxis and the ibidi Stage Top Incubator were applied in order to monitor directed cell migration under physiologic conditions. Within a time period of 24 hours, 145 pictures were taken in 10 minute intervals. These images serve as raw data for subsequent cell tracking.

Live cell imaging using the ibidi Stage Top Incubator shows migration of MDA-MB-231 cells in a chemotaxis assay. Phase contrast in a µ-Slide Chemotaxis; 4x objective lens.

Visualization of the Contraction of Cardiomyocytes

For the visualization of the contraction rates of cardiomyocytes, the actin cytoskeleton of iPSC-derived cardiomyocytes was stained using LifeAct. Sixteen hours after the transfer, the contractions per minute were measured. The myocytes showed contraction rates of about 70 beats per minute.

High-resolution live cell imaging for the visualization of the contraction rates of cardiomyocytes. F-Actin is stained with LifeAct. 100x objective lens.

Live Cell Imaging of Actin Dynamics in a Chemotactic Gradient

F-actin networks play an important role during cell migration, which can be investigated in detail using chemotactic gradients. Primary dendritic cells were isolated from mice and stained using LifeAct.

For the chemotaxis assay, cells were seeded on the µ-Slide Chemotaxis and a chemotactic gradient (CCL19) was applied. One day after the transfection, F-actin dynamics in the migrating cells were visualized using live cell imaging.

Live cell imaging of actin dynamics in a LifeAct-expressing primary dendritic mouse cell after the application of a chemotactic gradient.

Imaging of Actin Dynamics Under Flow

Several cell types in biofluidic vessels, such as endothelial cells and immune cells, are constantly exposed to shear stress in vivo. This mechanical stimulus has a great impact on the physiological behavior and adhesion properties of cells, and should be taken into account when performing respective studies.

With help of the ibidi channel slides, µ-Slide I Luer or µ-Slide VI ^0.4, and the ibidi Pump System, the F-actin cytoskeleton can be visualized in LifeAct-expressing, living cells under shear stress conditions. The ibidi Pump System is ideal for the long-term application of physiological shear stress to a cell layer and enables the adjustment of different flow rates. The system is fully compatible with live cell imaging and high-resolution fluorescence microscopy. Optionally, the fixation and immunofluorescence staining of the cells can be directly performed in the µ-Slide I Luer.

Experimental Setup:

• Device: ibidi Pump System
• Slide: µ-Slide I ^0.4 Luer (ibiTreat)
• Cells: LifeAct-expressing endothelial cells (HUVEC, P1)
• Shear stress parameters: 20 dyn/cm²

Live cell imaging under flow: actin cytoskeleton visualization in HUVEC after transduction with a LifeAct Adenoviral Vector and cultivation under 20 dyn/cm².