The Power of Duolink® flowPLA Detection Kits in Studying Protein Interactions, Post-Translational Modifications, and Low-Abundant Protein Targets

Duolink® PLA technology has been used by researchers to study protein events via microscopy and more recently in flow cytometry experiments. This article describes the new possibilities that are available to flow cytometry users to study protein-protein interactions, post-translational modifications, and low-abundant targets.

Figure 1 shows the basic steps to perform a Duolink® assay for flow cytometry using a Duolink® flowPLA Detection Kit. During the first three steps, the cells are prepared for the Duolink® assay. Single-cell suspensions are pre-treated with respect to fixation and permeabilization and then blocked in bulk solution (Step 1). Cells are then aliquoted in tubes or 96-well plates (Step 2) and incubated with primary antibodies (Step 3). The Duolink® PLA portion of the procedure  includes the incubation with the PLUS and MINUS PLA probes (Step 4), incubation with ligation solution (Step 5), incubation with amplification solution (Step 6), and finally, incubation with detection solution (Step 7) with washing between each step. Once the Duolink® PLA procedure is complete, the amount of PLA signal in individual cells of a cell population is measured by flow cytometry (Step 8) and the data analyzed (Step 9).

 

Figure 1. Duolink® PLA Protocol for Flow Cytometry Detection

Preparation Fix cells, permeabilize and block as bulk suspension Aliquot cells in tubes or 96-well plates (+/- filters) Add primary antibodies
Step 1: Fix cells, permeabilize and block as bulk suspension
Step 2: Aliquot cells in tubes or 96-well plates (+/- filters)
Step 3: Add primary
antibodies
DuoLink PLA Wash and add the PLUS and MINUS PLA probes Wash and add the ligation solution Wash and add the amplification solution Wash and add the detection solution
Step 4: Wash and add the PLUS and MINUS PLA probes Step 5: Wash and add the ligation solution Step 6: Wash and add the amplification solution Step 7: Wash and add the detection solution
Analysis Run flow cytometry Data analysis
Step 8: Run flow cytometry Step 9: Data analysis

 

The Duolink® PLA Flow Cytometry Protocol is Agnostic of Flow Cytometry Instrument

The same Duolink® assay performed using the Duolink® flowPLA Detection Kit – FarRed to detect a protein-protein interaction was analyzed using multiple flow cytometers. Data in Figure 2 show results from the Miltenyi Biotec MACSQuant® and the Amnis IMAGEStreamX® instruments, but comparable results were also achieved using the Guava® easyCyte 8HT and the BD FACSAria™ systems.

 

ADuolink® PLA flow cytometry kit with read-out on the MACSQuant® with FlowJo® analysis (A) BDuolink® PLA flow cytometry kit with read-out on the ImageStreamX® (B)
Key Sample ID
PLA with O/N amplification
PLA with 4 hr amplification
PLA with 100 min amplification
No primary antibodies control, O/N amplification
Unstained cells

Figure 2. Duolink® PLA analyzed using multiple flow cytometers produce comparable results.

Results show typical histogram from multiple flow cytometers illustrating reproducibility and flexibility of the protocol with respect to instrumentation. A protein-protein interaction was queried via the Duolink® PLA flow cytometry kit with read-out on both the MACSQuant® with FlowJo® analysis (A) and the ImageStreamX® (B). The Cy5 filter set was used on both instruments. Note: Some background staining was observed in the negative control compared to unstained cells, but the PLA signal intensity was significantly greater in the experimental samples that had undergone a Duolink® proximity ligation assay.

Detection of a Low-Abundant Epigenetic Protein Post-Translational Modification Using a Duolink® flowPLA Detection Kit

Epigenetics (heritable changes in gene expression that occur without alteration in DNA sequence) is an important factor in cancer development and works by altering the regulation of genes involved in cell growth, survival, or differentiation through histone modifications. Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that catalyzes the trimethlyation of lysine 27 of histone H3. This modification leads to transcriptional repression and is prevalent in many types of human cancers, such as liver, breast, and prostate cancer. Prior to the advent of Duolink® PLA technology, the interaction between EZH2 and histone H3 at K27 would have been difficult to detect by microscopy due to the transient nature of the interaction. To query this interaction in large cell populations by traditional flow cytometry would be impossible as flow cytometry can only measure co-expression levels, not protein interactions, and the low abundant nature of an epigenetic marker would make it challenging to detect.

To showcase the power of Duolink® PLA technology in studying protein interactions, post-translational modifications, and low-abundant targets via flow cytometry, the EZH2/H3K27me3 interaction was queried. DU145 human prostate cancer cells were grown on a chambered slide, fixed, permeabilized, and blocked for analysis by microscopy. Alternatively, the DU145 cells were detached, then fixed, permeabilized, blocked in suspension, and aliquoted into 96-well plates for analysis by flow cytometry. The Duolink® assay was performed using rabbit anti-H3K27me3 (SAB800015) and mouse anti-EZH2 (415M-156) primary antibodies, anti-rabbit PLUS and anti-mouse MINUS PLA probes, and the Duolink® flowPLA Detection Kit – FarRed, according to the Duolink® PLA Flow Cytometry Protocol. Technical negative controls included incubation with each primary antibody separately and no primary antibody. A schematic of this Duolink® PLA reaction is shown in Figure 3D.

A few PLA signals, as seen as red punctate spots, were detected in the nuclei of DU145 cells by fluorescence microscopy (Figure 3A), showing the low abundance of the EZH2/H3K27me3 interaction. However, detecting this interaction by flow cytometry was difficult, as indicated by the slight shift in the Duolink® PLA signal intensity of the orange peak compared to the green peak in Figure 3B, presumably due to the low abundance of this interaction. By extending the Duolink® PLA amplification time from the standard 100 minutes to overnight, enhanced detection was achieved (Figure 3B, pink peak). Minimal PLA signal was detected in the single primary antibody negative controls. Of note, extending the amplification time using the original Duolink® PLA detection reagents resulted in higher background because the fluorescent-labeled detection oligonucleotides are supplied in the amplification buffer. In the Duolink® flowPLA Detection Kits, the fluorescent-labeled detection oligonucleotides are supplied in a separate detection buffer, allowing more flexibility in the amplification and detection times for the user to obtain optimal results.


Combining Duolink® PLA technology with imaging flow cytometry allows localization of proteins or protein events (interactions or modifications) in large cell populations. We next analyzed the EZH2/H3K27me3 interaction using the Amnis ImageStream X® (Figure 3C). Discrete Duolink® PLA signals were detected in cells when the amplification incubation was the standard 100 minutes. However, the extension of the amplification time to overnight resulted in coalesced signals. Therefore, extended amplification time for low-abundant targets can cause coalescence of the individual PLA signals and may be unnecessary for imaging flow cytometry due to the exquisite sensitivity of the optics of the system.

A Few PLA signals (red) in the nuclei (blue) of DU145 cells were detected by fluorescence microscopy after 100 min amplification. BExtended amplification times enhanced the detection of EZH2-H3K27me3 interactions by conventional flow cytometry
Key Sample ID
EZH2/H3K27me3 PLA,
O/N amplification
EZH2/H3K27me3 PLA,
100 min amplification
Anti-EZH2 antibody only control, O/N amplification
Anti- H3K27me3 antibody only control, O/N amplification
No primary antibodies control, O/N amplification
 No primary Ab EZH2/H3K27me3 PLA, 100 min amplification EZH2/H3K27me3 PLA,
O/N amplification
 
CAnalysis of the EZH2-H3K27me3 interactions by imaging flow cytometry. DSchematic of the Duolink PLA reaction.

Figure 3. Increased amplification time during Duolink® PLA can aid in the detection of low-abundant protein targets by flow cytometry.

Duolink® PLA was performed to detect the trimethylation of lysine 27 on histone 3 (H3K27me3) mediated by EZH2. A) Few PLA signals (red) in the nuclei (blue) of DU145 cells were detected by fluorescence microscopy after 100 min amplification. FITC- Phalloidin-stained actin (green) was used as a counterstain. B) Extended amplification times enhanced the detection of EZH2-H3K27me3 interactions by conventional flow cytometry. C) Analysis of the EZH2-H3K27me3 interactions by imaging flow cytometry provided localization, but extended amplification causes coalescence of the PLA signals. D) Schematic of the Duolink PLA reaction.

In summary, the data presented here demonstrates the power of Duolink® PLA technology as a tool that can enable the use of flow cytometry to query protein-protein interactions, post-translational modifications, and low- abundant protein events. The flexibility of the technology allows the use of a multitude of flow cytometry instrumentation with the appropriate filter sets. The ability to use flow cytometry as a read-out for these types of interactions provides an exciting new avenue for protein detection.

 

Materials

     

References

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