Troubleshooting Guide for Duolink^® PLA for Flow Cytometry Detection

The combination of Duolink^® PLA technology and flow cytometry allows quantitative detection of protein-protein interactions, post-translational modifications, and protein expression with greater statistical power. In order to get the best results, a lot of planning, prep work, and troubleshooting is required. This guide provides general tips and tricks for proper experiment execution, aids in identifying potential problems, and provides solutions to ensure a successful Duolink^® PLA experiment for flow cytometry.

General Tips & Tricks

• Upfront Considerations
• Common Experimental Parameters
• Storage Conditions
• Data Analysis

Troubleshooting

• Too High of Background Signal
• Low or Lack of Signal
• Low Cell Retention
• Data Acquisition

Frequently Asked Questions

• General Questions
• Protocol and Analysis
• Specific Requirements
• Duolink^® Probemaker

Tip & Tricks

This section highlights general considerations and delineates important reminders to help your Duolink^® PLA experiment for flow cytometry proceed smoothly.

Upfront Considerations

• Use conditions suitable for optimal primary antibody performance within the sample to be tested. These include sample processing parameters (fixation and permeabilization), primary antibody titer, and primary antibody incubation conditions (temperature and duration). When two primary antibodies are used for a Duolink^® PLA experiment, make sure the sample processing conditions are compatible for optimal performance of both primary antibodies.
• Titration of primary antibody should be determined first by immunofluorescence (IF) by traditional flow cytometry (preferable) or microscopy. It is recommended to use indirect detection during initial optimization as directly conjugated antibodies may not represent the appropriate amount of antibody needed. When done properly, minimal additional optimization is required when applied to Duolink^® PLA technology.
• Duolink^® Blocking Solution and Antibody Diluent have been optimized for use with Duolink^® PLA reagents to minimize non-specific binding of the PLA probes and detection oligos. If alternative solutions have been optimized for primary antibody performance by traditional flow cytometry, it is likely these can be used in a Duolink^® PLA experiment but will need to be determined by the user.
• If combining Duolink^® PLA technology with IF for multicolor flow cytometry, choose fluorophores that minimize spectral overlap and reserve the brightest fluorophore option for the lowest expressing signal.
• Always include both technical and, when possible, biological controls in your experiment to properly evaluate the results.

Common Experimental Parameters

• Make sure to remove excess wash solutions from samples. Residual wash buffer can cause further dilution of antibodies and/or decrease ligation or amplification efficiency.
• Perform all steps at the appropriate temperatures and incubation times for best results, in particular the enzymatic steps (ligation and amplification).
• For detection of low-abundant proteins, extended amplification and/or detection times may be required.
• Perform washes in ample wash buffer at room temperature.
• Make sure ligation, amplification, and detection buffers are completely thawed and vortexed prior to usage.

Storage Conditions

• All Duolink^® PLA probes must be stored at 2 - 8°C; this includes those generated using Duolink^® Probemaker (PLUS or MINUS). Do not freeze PLA probes or results will significantly decrease.
• Store all Duolink^® flowPLA Detection Reagents at -20°C. Keep enzymes in a freezer block while in use.
• Do not store diluted Duolink^® PLA reagents.
• Resuspend cells in 1x PBS following the final wash step prior to analysis. Cells can be analyzed immediately or stored in the dark at 2 - 8°C for up to a week.

Data Analysis

• Duolink^® flowPLA detection reagents should be selected based on the lasers and detectors in your instrument. Duolink^® flowPLA fluorophore specifications are shown in Table 1.
• If combining Duolink^® PLA technology with IF for multicolor flow cytometry, choose fluorophores that minimize spectral overlap and reserve the brightest fluorophore option for the lowest expressing signal.
• Use forward and side scatter parameters to gate on fixed cells and to remove cellular debris.
• Be sure to collect enough events during analysis, especially in cases of rare events, to get significance and an accurate population spread.
• Properly gate on controls to ensure correct positive signal.

*The excitation laser line represents commonly used lasers that excite the fluorophore. It does not necessarily reflect the lasers available for each particular instrument.

Troubleshooting

Despite all precautions, sometimes the results are not as expected. Lack of signal in a positive control or high background are two of the most common issues when performing immunodetection experiments, and may also occur with the Duolink^® PLA technology. The tables below list the probable causes and suggested solutions to get around these common problems.

Too High of Background Signal

Low or Lack of Signal

Cell Retention

Data Acquisition

Frequently Asked Questions

Refer to the Duolink^® PLA Troubleshooting Guide for a list of frequently asked questions around the Duolink^® PLA technology.