Fluorescent proteins provide dynamic and spatial information at the level of cells, tissues, and organisms, and have enabled tremendous insights in biological research over the past several decades. Traditional methods for using fluorescent proteins are constrained by low expression efficiency and cytotoxicity associated with lipid-based transfection, and the accessibility of these tools has been limited to researchers skilled in molecular biology. The baculovirus-based CellLight® reagents are rapidly transforming fluorescent protein–based live-cell imaging from the domain of a handful of experts into a simple and powerful technique available to everyone.
Powered by the BacMam 2.0 platform, CellLight® reagents offer a drastically simplified workflow, high efficiency of transduction in a broad range of cell types, and exquisite intracellular targeting to organelles and other subcellular structures. These reagents are compatible with traditional fluorescent reagents—for multiplex labeling using a wide array of fluorescent probes—ultimately enabling a deeper understanding of biological processes within the context of living, functioning cells (Figure 1) or fixed and permeabilized cells.
Figure 1. Imaging primary cells with CellLight® reagents. Primary human skeletal muscle cells (GIBCO® HSkM-S cells) were cultured in poly-D-lysine–coated glass-bottomed petri dishes and transduced with CellLight® ER-GFP and CellLight® Golgi-RFP in complete medium to label the endoplasmic reticulum (green) and the Golgi apparatus (magenta), respectively. The following day, the cells were loaded with 1 µg/mL Hoechst 33342 (to label nuclei, blue) and 25 nM MitoTracker® Deep Red (to label mitochondria, red) for 5 min at 37°C in DPBS. Cells were washed three times and imaged using standard DAPI/FITC/TRITC/Cy®5 filters.
How CellLight® Reagents Work
|CellLight® Reagent|| |
|Histones (Histone 2B)|
|BacMam 2.0 Transduction Control|
|Null virus (control)|
* Excitation and emission maxima, in nm. † BacMam 1.0.
Amazing Images, Amazingly Simple Workflow
Figure 2. Simple one-step gene delivery with BacMam 2.0 reagents.
Figure 3. Imaging stem cell–derived cardiomyocytes with CellLight® reagents. Cardiomyocytes were incubated overnight with CellLight® Actin-GFP and CellLight® Mitochondria-RFP in complete medium. Cells were stained the following day with Hoechst 33342, then imaged using standard DAPI/FITC/TRITC filters. The striations of actin filaments characteristic of muscle cells are clearly evident.
Better Transduction of More Cell Types
Figure 4. Live-cell imaging of neurons using CellLight® Tubulin-RFP. Hippocampal tissue from postnatal day 3 rats was harvested and dissociated in neural culture medium, then plated onto glial feeder cultures. Cells were resuspended at 50,000 cells/mL in complete neural culture medium plus mitotic inhibitors. The medium was removed from the feeder cultures and replaced with 2 mL of the neural cell suspension. CellLight® Tubulin-RFP (50 µL) was added to the plates, and cells were incubated overnight. Fluorescence was observed in neurons after 48 hr in culture.
Control Reagents for Optimizing Fluorescent Protein Expression
Lights. Cells. Action.
- Kost TA, Condreay JP, Jarvis DL (2005) Nat Biotechnol 23:567–575.
- Fornwald JA, Lu Q, Wang D et al. (2007) Methods Mol Biol 388:95–114.
- Kaneko H, Suzuki H, Abe T et al. (2006) Biochem Biophys Res Commun 349:1220–1227.
- Hirasawa T, Yoshikawa K, Nakakura Y et al. (2007) J Biotechnol 131:34–44.
- Zeng J, Du J, Lin J et al. (2009) Mol Ther 17:1585–1593.
- Learn More about CellLight® Reagents