Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed
© Zaitoun et al; licensee BioMed Central Ltd. 2010
Received: 28 July 2010
Accepted: 6 December 2010
Published: 6 December 2010
Flow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins. The normal conformational structure of fluorescent proteins must be maintained in order to fluoresce. Whether or not RNAlater would affect signals from different types of intrinsic fluorescent proteins is crucial to its use in flow cytometry; this question has not been investigated in detail.
To address this question, we analyzed the effect of RNAlater on fluorescence intensity of GFP, YFP, DsRed and small fluorescent molecules attached to secondary antibodies (Cy2 and Texas-Red) when used in flow cytometry. FACS results were confirmed with fluorescence microscopy. Our results showed that exposure of YFP and GFP containing cells to RNAlater reduces the intensity of their fluorescence to such an extent that separation of such labeled cells is difficult if not impossible. In contrast, signals from DsRed2, Cy2 and Texas-Red were not affected by RNAlater treatment. In addition, the background fluorescence and clumping of dissociated cells are altered by RNAlater treatment.
When considering gene expression studies using cell sorting with RNAlater, DsRed is the fluorescent protein of choice while GFP/YFP have severe limitations because of their reduced fluorescence. It is necessary to examine the effects of RNAlater on signals from fluorescent markers and the physical properties (e.g., clumping) of the cells before considering its use in cell sorting.
Fluorescent labeling enables researchers to trace optically a particular population of cells in vitro or in vivo. FACS procedure is used to separate targeted populations for further biochemical characterization and in particular to permit isolation of intact mRNA for microarray and quantitative real time PCR studies. However, sorted cells go through a series of steps that could induce stress and change gene expression. Mechanical force has been shown to modulate global gene expression and signaling pathways in different cell types [1, 2]. Such force is typically used in dissociating cells. The hydrodynamic forces utilized in the operation of the FACS could affect cell viability as well. Indeed, several reports have shown a significant decrease in viability in different cell types after sorting by flow cytometry [3–7]. We observed a reduction of ~10% in the viability of sorted cells, which is consistent with these reports. While FACS is an efficient method for isolating cells for gene expression analysis, it is essential to prevent changes in normal global gene expression of sorted cells, a result which can be effected by treating cells with RNAlater. RNAlater preserves the product of normal gene expression by denaturing RNase and other cellular proteins, thus maintaining RNA integrity for gene expression studies using both microarray and quantitative real time PCR [8, 9]. RNAlater contains ammonium sulfate salt solutions, which have the ability to denature RNase at a controlled pH [10, 11]. However, if cells are prepared in RNAlater prior to sorting, the conformational structure of fluorescent proteins must be maintained within certain limits in order to fluoresce [12–14]. Because of its ability to denature protein, we investigated whether RNAlater would affect signals from fluorescent markers, such as GFP, YFP, DsRed, Cy2 and Texas-Red.
Results and Discussion
Our findings indicate that RNAlater treatment reduces GFP and YFP fluorescence, making separation of fluorescent and non-fluorescent cells difficult or impossible. Rosenberg et al. (2003) found a similar decrease in GFP fluorescence but they did not attempt to separate the two populations using flow cytometry. In addition, their results in tissues indicated that fluorescence from both GFP and DsRed was stable over an extended period of time. In contrast, we show that GFP fluorescence was extinguished in minutes, while DsRed fluorescence was stable. Their experiments were conducted using a GFP expressing cell line that was transfected with DsRed. One possible explanation for this difference in stability of fluorescence in tissues is that they might have detected the green fluorescence from the monomeric immature species of DsRed protein , and not from GFP itself. Indeed, we found that the green fluorescence from immature species of DsRed2 to be more resistant to quenching by RNAlater (pH 4.0) than red fluorescence from mature DsRed2.
It is noteworthy that RNAlater affects the physical properties of treated cells. Because of their large size, the changes (clumping) in the COS-7 and HEK 293 cells were more apparent than changes in primary cells. As a result, the sorter recognized the majority of RNAlater-treated COS-7 and HEK 293 cells as debris, while a large proportion of primary cells remained as singlets and were sorted normally.
In summary, we report that RNAlater diminishes the intensity of the fluorescent proteins GFP and YFP, and hinders their utility for sorting by flow cytometry. In contrast, RNAlater does not diminish the fluorescence of DsRed2 protein and the small molecule fluorophores Cy2 and Texas-Red. These results suggest that targeted DsRed2 expression in mice should be the choice for gene expression studies when RNAlater is used. However, it should be noted that RNAlater affected the physical properties (clumping) of the cells and their ability to be sorted regardless of the fluorescent protein expressed. Thus, it is necessary to examine the effects of RNAlater on signals from fluorescent markers and the physical properties of the cells before considering its use in cell sorting.
All procedures were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison. We obtained tissues from transgenic mice that express YFP in neural crest-derived cells . Pregnant mice were anesthetized with isoflurane vapor, sacrificed by cervical dislocation, and fetuses were removed at E14.5. YFP positive fetuses were identified under the fluorescent microscope and their gastrointestinal tracts were harvested and pooled. Collected tissues were dissociated in a mixture of 3 mg/ml collagenase, 1 mg/ml Dispace, 1 mg/ml BSA, and 0.5 mg/ml DNAase for 20 minutes at 37°C, washed in PBS, and triturated. The dissociated cells were resuspended in either 0.1% bovine serum albumin in PBS (BSA) or RNAlater (Qiagen, Hilden, Germany, final concentration ~50% in BSA), and kept on ice for 0.5-1 hour before FACS sorting.
For whole mount staining, paraformaldehyde-fixed tissues were incubated with human anti-Hu (Epstein laboratory, Madison, WI), followed by goat anti-human-Texas Red (Jackson ImmunoResearch, West Grove, PA). Fixed dissociated cells were incubated with chicken anti-GFP (Aves, Tigard, Or) for 2 hours at room temperature, washed in PBS, incubated in donkey anti-chicken-CY2 (Jackson ImmunoResearch) for 2 hours, washed in PBS, and sorted on the flow cytometer as described below.
Cell culture and DsRed2 transfection
The GFP positive T cell line was cultured as described . HEK-293 cells were cultured in Eagle's minimum essential medium (Fisher Scientific, Pittsburg, PA) with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% L-glutamine, 1% sodium pyruvate, and 400 μg/ml gentamicin. COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Fisher Scientific) with 10% cosmic calf serum and 1% penicillin/streptomycin. Both cell types were cultured in 37°C 5% CO2-air atmosphere. HEK-293 cells and COS-7 cells were transfected with DsRed2 plasmid by electroporation as described previously . After 3-5 days of transfection, both lines were dissociated with trypsin into single cells and kept on ice for 0.5-1 hour. All three cell lines were sorted on the flow cytometer as described below.
Cell suspensions were prepared as described above. 0.25 ml of dissociated cells in BSA was filtered through 20 um Nitex into tubes containing either 1.0 ml RNALater or 1.0 ml BSA. Filtered cells were separated into fluorescent positive and negative cells by a flow cytometry (FACS Vantage SE, BD Bioscience, San Jose, California).
The authors are grateful to Dr. Christine Seroogy and Lauren Nettenstrom for providing the T cell line, Dr. Erik W. Dent for providing DsRed2 plasmid, Brian Torres and Dr. Arnold E. Ruoho for providing the COS-7 and HEK-293 cell lines and for assistance in DsRed2 transfection, and to the UWCCC flow cytometry staff. This work was supported by grant R01-DK081634 from the National Institutes of Health (M.L.E) and in part by NIH/NCI P30 CA014520-UW Comprehensive Cancer Center Support.
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