A versatile, bar-coded nuclear marker/reporter for live cell fluorescent and multiplexed high content imaging

Abstract

The screening of large numbers of compounds or siRNAs is a mainstay of both academic and pharmaceutical research. Most screens test those interventions against a single biochemical or cellular output whereas recording multiple complementary outputs may be more biologically relevant. High throughput, multi-channel fluorescence microscopy permits multiple outputs to be quantified in specific cellular subcompartments. However, the number of distinct fluorescent outputs available remains limited. Here, we describe a cellular bar-code technology in which multiple cell-based assays are combined in one well after which each assay is distinguished by fluorescence microscopy. The technology uses the unique fluorescent properties of assay-specific markers comprised of distinct combinations of different 'red' fluorescent proteins sandwiched around a nuclear localization signal. The bar-code markers are excited by a common wavelength of light but distinguished ratiometrically by their differing relative fluorescence in two emission channels. Targeting the bar-code to cell nuclei enables individual cells expressing distinguishable markers to be readily separated by standard image analysis programs. We validated the method by showing that the unique responses of different cell-based assays to specific drugs are retained when three assays are co-plated and separated by the bar-code. Based upon those studies, we discuss a roadmap in which even more assays may be combined in a well. The ability to analyze multiple assays simultaneously will enable screens that better identify, characterize and distinguish hits according to multiple biologically or clinically relevant criteria. These capabilities also enable the re-creation of complex mixtures of cell types that is emerging as a central area of interest in many fields.

Figure 1. Construction and utility of the FP_NLSFP nuclear marker.

A, Amino acids inserted (black font, SV40 NLS underlined) between two mCherry FPs (orange font) within the mCherry_NLSmCherry nuclear marker. The insertion sequence and location is similar for the other FP_NLSFP nuclear markers created in this study. B, Nuclear fluorescence of the mCherry_NLSmCherry marker stably expressed in a HeLa cell line in relationship to nuclei stained with Hoechst 33342. The locations of a YFP-tagged Androgen Receptor (AR) co-expressed in this cell line also are shown. The cells were grown in media containing testosterone, which translocates the AR into the cell nuclei. Images were captured with a 10x objective. *, mitotic cell. #, dying cell.

Figure 2. Segmentation of cell nuclei marked with FP_NLSFP.

The boundaries of objects with contiguous FP_NLSFP expression established by a commercial analysis software (yellow circles) marked the boundaries of nuclei stained with the DNA binding dye Hoechst 33342. *, colony of cells in which FP_NLSFP expression is lost sporadically.

Figure 3. Improved well-to-well reproducibility in cell growth measurement enabled by the FP_NLSFP live cell nuclear marker.

Variations in cell numbers plated in each well (Day 0) obscured the ability to reliably detect an increase in cell number after four days of slow growth by LNCaP-C4-2 prostate cancer cells treated with vehicle or 0.2 nM DHT. Each symbol represents the numbers of A, Hoechst 33342-stained nuclei or B, FP_NLSFP-marked nuclei segmented in each well. C, Dividing the number of FP_NLSFP-marked cells on Day 4 by the baseline (Day 0) number of FP_NLSFP-marked cells in the same well improved the reproducibility of growth measurement. Dotted lines, three standard deviations (3sd) above the mean Day 0 (black dotted line) or Day 4 vehicle-treated (gray dotted line) measurements are shown. The 3sd cut-offs were used to determine the number of, respectively, vehicle-treated and DHT-treated wells that were scored falsely in the Day 0 and vehicle-treated wells.

Figure 4. Cellular “bar-coding”.

A, Excitation and emission properties of four FPs used to create the bar-code. All FPs were excited by light of 560–590 nm (orange box) but emitted different relative amounts in two emission channels (em1: 635–675 nm; em2: 608–648 nm). For example, the area under the curve collected for mPlum in em1 would be slightly more than that in em2 whereas, for mCherry, em2 emissions would be much higher than em1 emissions. These differences were seen in practice (Table 3). B, The measured em1/em2 ratios for sixteen FP_NLSFP markers using all possible combinations of the four FPs (Table 4) were similar to those predicted if one assumes no FRET amongst the FPs. The theoretical em1/em2 ratios were calculated from their relative abilities to be excited by 560–590 nm light (Fig. 4A), their relative brightness once excited (Table 3) and the em1 and em2 emissions detected by our instrument for the four homogeneous FP_NLSFPs (mPlum_NLSmPlum, mCherry_NLSmCherry etc).

Figure 5. Application of bar-code to cell counting studies.

A, Concept of bar-code for mixing differentially marked FP_NLSFP expressing cells. B, Differential response of two LNCaP-C4-2 cell subclones to an inhibitor of cell growth (actinomycin D). C, em1/em2 ratios of all cells within a representative well (x-axis) compared to the intensities of each cell in the em1 channel. D, LNCaP-C4-2 cells mixed, co-plated, treated exactly as in figure 5B then separated according to the bar-code showed similar treatment responses to the individually plated cells. Growth measurements are shown as the mean +/− sd from 8 (Fig. 5B) or 16 (Fig. 5D) wells for each treatment condition. *, statistically significant (p<0.01) increases or decrease in cell number relative to vehicle-treated cells; #, statistically significant (p<0.01) increase in cell number of DHT/actinomycin D treated wells relative to actinomycin D-treated wells.

Figure 6. Bar-code separation of different, co-cultured assays.

Representative images of A, the em1/em2 ratio and B, YFP-tagged AR showed the FP_NLSFP bar code to accurately discriminate between two differentially marked HeLa cell lines in which a wild-type (wt) or mutant (T877S) AR have different nuclear distributions when grown with 10⁻⁸ M estradiol. C, Quantification of nuclear AR levels in the two different cell lines after incubation with 10⁻⁷ M of the indicated steroids demonstrated that the differential responses between the wt and T877A ARs observed when plated separately were retained when the cell lines were mixed in a well and sorted according to the bar-code. Nuclear AR measurements are shown as the mean +/− sd from 48 wells for each treatment condition. The distinct responses of the two assays to different hormones are indicated by *, # (statistically significant increases, p<0.01, that were at least double the nuclear AR-YFP intensities in vehicle-treated mPlum_NLSmPlum and mCherry_NLSmCherry cells, respectively).

Figure 7. Characterization of three differentially bar-coded LNCaP-C4-2 cell lines, each expressing a different YFP-based reporter assay.

A–C, YFP fluorescence intensities within the FP_NLSFP-marked cell nuclei of each separately-cultured reporter line are shown in response to fifteen different steroids (10⁻⁸ M each). Nuclear YFP measurements are shown as the mean +/− sd from 10 fields for each treatment condition. Gray bars, measurements for indicated assay plated independently into separated wells. Black bars, measurements from wells in which the three assays are co-cultured and separated by the bar-code. The dotted gray line represents the measurements obtained upon treatment of the independent assays with vehicle only. Steroids: 1: pregnenolone, 2: progesterone, 3: 11-deoxycorticosterone, 4: aldosterone, 5: 17-hydroxypregnenolone, 6: 17-hydroxyprogesterone, 7: 11-deoxycortisol, 8: cortisol, 9: dehydroepiandrosterone, 10: androstendione, 11: estrone, 12:4-androstenediol, 13: testosterone, 14: estradiol, 15: dihydrotestosterone. veh, wells treated with vehicle only. *, steroids that increase an assay (p<0.05).

Figure 8. Distinct em1/em2 ratios characteristic for each of the three bar-coded LNCaP-C4-2 cell lines.

A–C, Data from a representative field for each cell line. Each ‘x’ represents a YFP (left panel) or em1 (next panel) fluorescent intensity measurement from a single cell in relationship to the em1/em2 ratio measured for that cell. The images from which the measurements were obtained are shown. The representative fields for A and C were from cells treated with an androgen while the fields shown for B were vehicle treated because the MMTV-YFP assay intensity is much higher upon androgen treatment. The em1/em2 ratios used to assign a specific cell to a specific bar-code are shown as colored bars on the y-axis. The margins for those characteristic em1/em2 ratios were established as 3 sd away for all cellular measurements for the independently grown assays.