2RiMED Foundation, Palermo, Italy
3Science of Transplantation PhD program, Pisa University, Pisa, Italy
*These authors contributed equally to this study
Intracellular staining is a widely used flow cytometry (FCM)-based technique to detect the expression of cytoslio nucleic antigens. However, intracellular staining of cells expressing cytosolic fluorescent protein (FP) markers was proven to be problematic as significant loss of the FP-signal was routinely observed. Using splenocytes harvested from mice constitutively expressing the enhanced yellow fluorescent proteins (YFP) as a model, we modified the widely used intracellular staining protocol and successfully achieved simultaneous detection of both the nuclar proteins and YFP in T-regulatory cells. The improved protocol can be used to perform antibody-based intracellular characterization of FP-labeled target cells, while maintaining their fluorescent reporter signals for easy tracing and identification.
Transgenic animals expressing a f luorescent protein (FP) in specific cell types or lineages are essential tools for studying biological processes in vivo (1). Frequently, to target a specific cell population, the promoter element governing the transcription of a tissue-specific gene is used to control FP expression directly or indirectly through the action of the Cre recombinase in a Rosa26-FP reporter line. Cre-mediated removal of the loxP-tagged transcriptional stopper cassette situated between the ubiquitously active Rosa26 promoter and the FP gene will switch on the FP gene transcription, resulting in permanent labeling of cells of the specific lineage with FP(3,4).
Nevertheless, it is essential to examine whether the promoter elements used to drive FP expression can truthfully recapitulate the tissue specificity of the endogenous gene in vivo (4). Staining FP-positive cells with antibodies specific to the endogenous proteins for flow cytometry (FCM) analysis is an effective way to validate the nature of the FP-labeled cells (5). In addition, antibody-based FCM can also facilitate the characterization of the molecular properties of the FP-labeled cells under various experimental conditions (6). However, FCM detection of cytosolic or nuclear proteins in FP-expressing cells has been shown to be technically challenging as loss of FP signal is frequently observed when the intracellular staining procedure is used. The common approach to overcome this obstacle is to first isolate the FP-expressing cells with the fluorescence activated cell sorter (FACS) technique, followed by intracellular characterization (7). Such an approach is not only time-consuming, but also impractical when studying rare cells.
In this study, we modified an intracellular staining protocol, which utilizes paraformaldehyde and saponin as the fixative and membrane permeabilizating reagent, respectively, to achieve simultaneous detection of nuclear proteins and cytosolic FP molecules. The nuclear Foxp3 proteins in T-regulatory cells of Vav-Cre:Rosa-YFP reporter mice (3) were used as a model target.
After staining YFP+ splenocytes with antibodies specific to surface markers of T cells (i.e., CD3 and CD4), we subjected them to the intracellular staining procedure outlined in Figure 1A (detailed in Supplementary material) for the detection of nuclear Foxp3 proteins. Although the Foxp3+ T-regulatory cell population could easily be identified, the YFP signals in these cells became essentially undetectable (Figure 1B). Loss of YFP signal could result either from fixative-induced conformation changes of the YFP proteins, or from the loss of cytosolic YFP proteins due to the leakiness of the permeabilized cell membrane. To investigate whether overfixation is the major causative factor, we shortened the Fixation/Permeabilization (Fix/Perm) buffer treatment from 2 hours to 5 minutes, but still failed to preserve detectable YFP signals (data not shown). Indeed, significant reduction of cellular YFP signal was observed immediately following exposure of YFP+ cells to the Fix/Perm buffer, indicating that the rapid loss of the fluorescent signal is due to diffusion of YFP molecules out of the permeabilized cells (Figure 1C and data not shown).
As fixation with 1%–2% paraformaldehyde is routinely used in FCM protocols to preserve fluorescent signals for analysis, we postulated that adding a fixation step prior to the Fix/Perm treatment might prevent YFP proteins from leaking out of the cytoplasmic membrane. Indeed, prefixing samples with 2% paraformaldehyde for 30 minutes can effectively retain the YFP signal; however, we then became unable to stain Foxp3 proteins (Figure 1D), presumably due to overfixation of the cytoplasmic membrane. We thus speculated that the success of staining the nuclear FoxP3 molecules, while preserving detectable YFP signals, might hinge on the optimization of pre-fixation of cytoplasmic membrane: to block the leakage of YFP molecules from cytosol, while preserving the accessibility of the antibody to its nuclear target.
To find out the minimal fixative conditions which can be used to preserve the YFP signal, cells were prefixed with various concentrations of fixative for different lengths of time (Supplementary Table 1), followed by Fix/Perm treatment and FCM analysis. As summarized in Figure 2A, the success of cytoplasmic YFP protein retention was determined by the combined effect of prefixation time and concentration of the fixative. To give a relative quantification of the prefixation process, we defined the value of prefixation time (minutes) x % of fixative as prefixation factor (PF), and concluded that conditions with PF≥4 (gray area in Fig ure 2A) were sufficient to retain enough YFP proteins for FCM analysis.
To evaluate the negative effect of prefixation on Foxp3 staining, we took advantage of the eGFP-Foxp3 transgenic mice, in which an eGFP molecule was inserted in-frame to the 5 ′ end of the foxp3 gene to encode a functional eGFP-Foxp3 fusion protein (8). One of the major functional components in the permeabilization buffer is saponin, which can complex with cholesterol to form pores in the cholesterol-rich cytoplasm membrane but leaves the cholesterol-poor nuclear membrane largely intact (9,10). Thus, nuclear eGFP-Foxp3 fusion protein should be retained in the nucleus during intracellular staining while the eGFP signal will truthfully reflect the presence of Foxp3 protein (Figure 2B). Splenocytes harvested from the eGFP-Foxp3 transgenic mice were prefixed with 2% paraformaldehyde from 15 seconds to 5 minutes, prior to staining of the cells with anti-Foxp3 antibodies. Prefixation from 15 seconds to 2 minutes did not drastically affect the percentage of Foxp3+ cells, whereas we began to observe a decrease of the antibody staining of the eGFP-Foxp3 fusion proteins after the cells were treated with fixative for more than 5 min (Figure 2B and data not shown).
Based on the above findings, we proceeded to add the prefixation step to the intracellular staining protocol for Foxp3 detection in Vav-Cre: RosaYFP T regulatory cells. As shown in Figure 2C, a distinct population of CD4+ T cells can be readily identified as double positive for both YFP and Foxp3 under a number of amenable conditions. Since various intracellular antigens may differ substantially in their retention and susceptibility to Fix/Perm treatment, we also examined the nuclear expression of helios, a member of the Ikaros family of nuclear expressed transcription factors, in YFP+ splenocytes (11). As shown in Supplementary Figure S1, effective detection of both Helios and YFP were achieved under conditions similar to those described in Figure 2C. Thus, PFs range from 4 to 20 represents the optimal conditions to obtain efficient nuclear protein staining, and well separation of the YFP signals from the background. It is conceivable that the optimal PF will vary in different models; for cells with weak fluorescent signals, a higher PF (10–20) could be preferred, and vice versa. Nevertheless, the simple modification of the intracellular staining procedure described above (Figure 2D) reliably enables the usage of cytoplasmic FPs as cellular markers for co-localization studies with FCM technology.
We thank Dr. Joy Williams for helpful discussions and critical reading of the manuscript. We also thank Mr. Carl Engman, Dr. Brett Philips and Dr. Valentina DiCaro for technical assistance. The study was supported in part by Department of Defense grant W81XWH-09-1-0742.
The authors declare no competing interests.
Address correspondence to Yong Fan, Ph.D., Rangos Research Center, Children's Hospital of Pittsburgh, Pittsburgh, PA, USA. Email: [email protected]
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