Obtain strong signals in cells with low transfection efficiency
by Jae Choi, Ph.D.; Janaki Narahari, Ph.D.; Kevin Albers, M.S.; Megan Dobbs, B.S.; Douglas Hughes, Ph.D.; Brian Webb, Ph.D. - 06/13/12
The plasmid-based luciferase reporter assay is a powerful tool for studying signal transduction pathways in mammalian cells. However, cells with low transfection efficiency, resulting in low luminescent signal output, can make detection difficult. The Thermo Scientific Pierce Gaussia, Cypridina, and Green Renilla Luciferase Reporter Assays produce a robust bioluminescent signal, enabling luciferase-based pathway studies in cells that are difficult to transfect.
In this study, we demonstrated the low transfection efficiency of Jurkat cells, which corroborated with another report (1). We then used the Pierce Luciferase Reporter Systems to transfect the cells and the Thermo Scientific Pierce Luciferase Flash Assay Kits to measure activity. We also constructed cAMP response element (CRE) reporter plasmids by cloning the CRE element plus minimal promoter into the Pierce pMCS-Luc vector and then activated the pathway in transfected cells. The highly sensitive Pierce Luciferase Reporter Systems were effective for measuring reporter activity in difficult-to-transfect cells.
Using the Thermo Scientific TurboFect Transfection Reagent (Part No. R0533), we transfected Jurkat cells with green fluorescent protein (GFP) plasmids and assessed transfection efficiency. Few cells (< 100 cells) exhibited GFP signals via fluorescent microscopy (Figure 1A). Quantification of transfection by fluorescence-activated cell sorting (FACS) revealed that total GFP-expressing cells were less than 1% (Figure 1B), which demonstrates the low transfection efficiency of Jurkat cells.
To demonstrate that a highly sensitive assay can compensate for cells with low transfection efficiency, we used the Pierce pCMV Gaussia, Cypridina, and Green Renilla luciferase reporter plasmids to transfect Jurkat cells and measured activity using the Thermo Scientific Pierce Luciferase Flash Assay Kits. The secreted Gaussia and intracellular green Renilla luciferase activities produced the strongest luciferase signals. Secreted and intracellular Cypridina luciferase also produced robust signals. Furthermore, our kit outperformed a kit from another supplier (Figure 2).
We also tested cAMP response element (CRE) promoter activity in Jurkat cells using the CRE-Gaussia, Cypridina, and Green Renilla reporter plasmids. After transfection (24 hours), we treated the cells with forskolin, a CRE activator, and measured the resulting luciferase activity using the Pierce Luciferase Flash Assay Kits. The results demonstrated that cAMP cellular signal pathway can be analyzed in Jurkat cells using the Pierce Luciferase Reporters. We also evaluated CRE-Firefly (Promega Inc.), which resulted in minimal signal (Figure 3).
Traditionally, successfully performing luciferase reporter assays in cells with low-transfection efficiency has been a challenge. The Pierce Luciferase Reporter Systems, however, have strong signal outputs, which enable signal transfection pathway analyses in cells with a transfection efficiency of less than 1%.
Cell Culture: Jurkat cells (clone E6-1, ATCC, TIB-152) were cultured at 37°C with 5% CO2 in RPMI-1640 with 10% fetal bovine serum with 1% penicillin/streptomycin. Cells were passaged every two days and maintained at 50-70% confluence. Log-phase cells were plated in 96-well plates for 6 hours before transfection to ensure that they were healthy.
Transfection: In each well of a 96-well plate, 150K log-phase Jurkat cells were seeded in 0.1mL of RPMI-1640 media 6 hours before transfection. Plasmid DNA (100ng) was diluted in 10μL of serum-free media. TurboFect Transfection Reagent (0.3μL; Part No. R0533) was added to the diluted DNA and mixed by pipetting. The mixture was added drop-wise to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours before any treatment.
Fluorescence microscopic analysis: Log-phase Jurkat cells were seeded in a well of a 96-well plate and incubated for 6 hours at 37°C in 5% CO2. pCMV-GFP plasmids (100ng/well) were added with TurboFect Transfection Reagent for 24 hours. The plate was briefly centrifuged to force down the cells. Images were acquired with a Nikon Eclipse TS100 using a LWD 40X/0.55 ph1 ADL objective. The fluorescence (GFP) image was overlaid with a bright-field image.
FACS acquisition and analysis: The BD FACScan Flow Cytometer (Becton Dickinson) was calibrated with Calibrate 3 Color Beads (Becton Dickinson), using FACSComp 4.2., under lyse/wash conditions. After passing calibration, the samples were processed and 10,000 events collected. Analysis was based on the detection of GFP similar to FITC with a photomultiplier tube detecting the emission at 530nm. Software (CellQuest Pro version 4.0.2, Becton Dickinson) was used to generate a histogram of cell counts vs. FLH (fluorescence intensity).
Luciferase reporter assays: 24 hours after transfection, the cells were briefly centrifuged (1000 × g, 3 minutes) to force cells to the bottom of the plate. The media was collected to measure activity. The cells were lysed with 100μL of Pierce Luciferase Cell Lysis Buffer (Part No. 16189). Activity in the media and lysates was determined using the appropriate luciferase flash assay kit: Gaussia (Part No. 16158); Cypridina (Part No. 16168); Green Renilla (Part No. 16164). The firefly activity was determined using the Bright-Glo Assay Kit (Promega, Inc.). Bioluminescence signals (RLUs) were detected using a Thermo Scientific Varioskan Flash Luminometer equipped with reagent injectors (Signal integration time = 1 second).
The CRE-Gaussia, -Cypridina and -Green Renilla Luc reporter plasmids, constructed by cloning CRE element plus minimal promoter (TATA box and ~40bp spacer before the Kozak sequence) into the Pierce promoterless pMCS-Luciferase Reporter Plasmids (Part No. 16146)(Part No. 16149)(Part No. 16152) were transfected into 150K Jurkat cells for 24 hours using the Pierce TurboFect Transfection Reagent (Part No. R0533). After transfection, cells were treated with forskolin for 5 hours to activate the CREB-dependent cAMP signal transduction pathway. The plate was briefly centrifuged (1000 × g, 3 minutes), and the media was carefully collected for activity measurement. The cells were lysed with Pierce Luciferase Cell Lysis Buffer (Part No. 16189). Activity in the media and lysates was determined using the appropriate luciferase flash assay kit: Gaussia (Part No. 16158); Cypridina (Part No. 16168); Green Renilla (Part No. 16164). The Firefly luciferase activity was determined using the Bright-Glo Assay Kit (Promega, Inc.). Bioluminescence signals (RLUs) were detected using a Varioskan Flash Luminometer equipped with reagent injectors (signal integration time = 1 second).
- Jordan, E.T., et al. (2008). Optimizing electroporation conditions in primary and other difficult-to-transfect cells. J Biolmol Tech 19(5):328-34.
Insel, P.A. and Ostrom, R.S. (2003). Forskolin as a tool for examining adenylyl cyclase expression, regulation, and G protein signaling. Cell Mol Neurobiol 23(3):305-14.
Mayr, B. and Montminy M. (2001). Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2(8):599-609.
Montminy, M.R., et al. (1986). Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci USA 83(18):6682-6.
Mayr, M.B., et al. (2005). Glutamine rich and basic region/leucine zipper (bZIP) domains stabilize cAMP response element binding protein (CREB) binding to chromatin. J Biol Chem 280(15):15103-10.
Preston, B.R. and Douillet, C. (2009). Bioluminescence: Methods and Protocols, Second Edition. Human Press.
Shimomura, O. (2006). Bioluminscence: Chemimal Principles and Methods. World Scientific Publishing Co. Ptc.Ltd.
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