pCMV/pCAG-LifeAct Plasmids

Applications:

• Cytoskeleton organization:
  - Non-toxic actin visualization
  - Fluorescence staining
• Cytoskeletal dynamics:
  - Actin dynamics in cellular processes
  - Live cell imaging and microscopy

Technical Features:

• For transient and stable transfections
• Superb biocompatibility - non-toxic staining of living samples

Specifications:

Storage	-20 °C
TagGFP2 (Exmax / Emmax)	483 / 506 nm
TagRFP (Exmax / Emmax)	555 / 584 nm

Watch our Movie:

Live Cell Imaging of Actin Dynamics in Primary Dendritic Mouse Cells, Transfected with p^CMVLifeAct-TagGFP2

Experimental Example: 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, the LifeAct Plasmid 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/.

LifeAct: A Versatile Tool for F-Actin Visualization in Living Cells

LifeAct, a 17-amino acid peptide, is derived from a protein from Saccharomyces cerevisiae. LifeAct stains filamentous actin (F-actin) structures in living or fixed eukaryotic cells and tissues. In contrast to GFP-actin and its alternatives such as actin-binding proteins, LifeAct does not interfere with actin dynamics in vitro and in vivo, as shown in several cell types and model organisms.*

LifeAct-Plasmids may be used in many cell types when a transient or stable expression of LifeAct is needed. In this case, researchers can choose between two different promoters, CMV (cytomegalovirus) or CAG (modified chicken ?-actin). For many standard cell lines, the CMV promoter is the best choice as it leads to strong expression of LifeAct. However, when working with sensitive cells such as neuronal or embryonic stem cells, it is most often recommended to use the CAG promoter, because it does not have a viral origin and an interference with cellular defense mechanisms ("silencing mechanisms") is less likely.

After transfection of cells with pLifeAct, F-actin is visualized using the fluorescence markers TagGFP2 or TagRFP**. Stable transfection of cell lines allows long-term actin staining for various applications.

Adenoviral vectors are available for difficult-to-transfect cells or when up to 100 % transgene expressing cells are needed. In this case, it is also possible to choose between the two promoters mentioned before.

In addition, different stable cell lines are available, which help facilitate daily lab life. You can immediately start performing experiments without the time-consuming and laborious generation of stable cell lines, while still assuring high-quality standards.

Method Comparison: Using LifeAct for F-Actin Visualization in Living Cells

	LifeAct-Plasmid With Torpedo DNA	LifeAct-mRNA With Fuse-It-mRNA	LifeAct-Protein With Fuse-It-P	LifeAct- Adenovirus	LifeAct- Lentivirus
Method Suitable for 1
Cell lines	+++	+++	+++	+++	+++
Primary cells	+	+++	++	+++	+++
Non-mammalian cells	-	+	+	-	-
Transfection Stability
Transient	Yes	Yes	Yes	Yes	No
Stable	Yes	No	No	No	Yes
Protocol and Analysis
Protocol simplicity	+++	++	++	+++	+++
LifeAct readout start	1-2 days	few hours	few minutes	1-2 days	1-2 days
LIfeAct readout duration	several days (infinity if stable)	several days	up to 1 day	several days	several days (infinity if stable)
Signal intensity	+++	+++	+	+++	+++
Extra Benefits
Prevention of overexpression artefacts	-	+	+++	+	+
Prevention of viral response	+++	+++	+++	-	-
Biocompatibility of transfer method	+	+++	+++	++	++
Actin functionality	+++	+++	+++	+++	+++
Safety Consideration
Biosafety Level	BSL1	-	-	BSL2	BSL2

¹ Not all of these combinations have been tested in all indicated cell types. We encourage you to use this table as a guideline to understand how the LifeAct system can be optimally applied.

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* Original LifeAct publication in Nature Methods:
Riedl J, et al. Lifeact - a versatile marker for the visualization of F-actin. Nature Methods 5, 605-607 (2008)

** Information about TagGFP2 and TagRFP at www.evrogen.com

LifeAct is a registered trademark of ibidi GmbH.

For license and product use information please refer to www.ibidi.com, Legal Notes.