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.

Figure 1. The Duolink PLA signal is stable. The same cells stored for a week give the same result as those immediately analyzed.

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.

Table 1. Duolink flowPLA Fluorophore Specifications

Product No.	Reagent	Excitation (nm)	Excitation Laser Line (nm)*	Emission (nm)
DUO94005	Duolink flowPLA Detection Violet	390	405	476
DUO94002	Duolink flowPLA Detection Green	495	488	527
DUO94003	Duolink flowPLA Detection Orange	554	532, 561	579
DUO94001	Duolink flowPLA Detection Red	594	532, 561	624
DUO94004	Duolink flowPLA Detection Far-Red	644	638, 640, 642	669

*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

Probable Causes	Suggested Solutions
Concentration of primary antibody is too high	Begin titration of each primary antibody using traditional indirect flow cytometry. Choose dilutions that achieve significant signal with minimal background. Include the use of each primary antibody alone during the Duolink® PLA experiment as technical controls as each may contribute differently to the background signal. Perform additional antibody titrations accordingly. If compensation is needed, ensure that signal intensity is optimal and spectral spillover is minimized.
Non-specific binding of primary antibodies	If background persists despite critical determination of assay conditions (fixation, permeabilization, and antibody titer), try an alternative primary antibody. If using immune cells, try adding 2.5-10 μg of an Fc blocker to the Blocking Solution to reduce antibody binding to Fc receptors. Use an isotype IgG from the same species as the primary antibodies to determine the level of non-specific background caused by the primary antibodies.
Insufficient blocking	Ensure the entire sample is covered in Blocking Solution. Increase blocking incubation time. Dilute the primary and PLA probes in the provided antibody diluent which contains blocking agents optimized for the Duolink® PLA technology. If background is high in the secondary control (no primary antibodies), try adding 15 µg/ml salmon sperm DNA to the detection buffer to decrease non-specific binding of the detection oligos.   Figure 2. Addition of salmon sperm DNA to the Duolink® flowPLA Detection Buffer can decrease background.
Insufficient washing	Increase number of washes, wash times, and/or wash volumes Use freshly made wash solutions Ensure sample is fully resuspended during wash steps If using an alternative wash buffer, add mild detergent (such as Tween®-20 or CHAPS) to buffer to increase stringency.
Drying of sample	Never let samples or filters (when using filter cups or filter plates) dry out after washes and before addition of reagents.
Precipitate in ligation buffer	Make sure ligase buffer is completely thawed and vortexed prior to using.
Dust, salt, or fixation precipitates cause highly fluorescent particles	Wash your cells at least twice to ensure that the culture medium is removed before adding the fixative. Use freshly made wash solutions. If the problem remains, sterile filter all washing solutions. Ensure samples are resuspended in an appropriate buffer (e.g., detergent-free PBS) for analysis.

Low or Lack of Signal

Probable Causes	Suggested Solutions
Inappropriate reaction volume	Knowledge of the abundance of the protein target or interaction can help determine the appropriate reaction volume. The recommended reaction volume for highly abundant interactions is 1µL per 1,000 cells (100,000 cells in 100 µL). Make the appropriate adjustment to the reaction volume as per cell number and/or protein target abundance. Ensure the reaction volume is sufficient to cover the entire sample. Figure 3. Varying the cell number per reaction can affect results.
No or insufficient binding of primary antibodies	Ensure sample processing conditions are optimal for primary antibody performance and location of target. When using two primary antibodies, sample processing needs to be compatible for both primary antibodies. Titrate each primary antibody separately using the single recognition PLA method.
Inappropriate dilutions of Duolink® PLA reagents	Duolink® flowPLA reagents are supplied as concentrated stocks. Dilute according to recommendations (1:5 for ligation, amplification, and detection buffers, 1:40 for ligase, and 1:80 for polymerase) Figure 4. Further dilution of Duolink® flowPLA reagents will adversely affect results.
Inappropriate storage of Duolink® PLA reagents	Follow the recommended storage conditions for all Duolink® PLA reagents. Do not store diluted Duolink® PLA reagents.
Inappropriate incubation temperatures	Perform all steps at the appropriate temperatures, in particular the enzymatic steps (ligation and amplification).
Inefficient ligation	Follow the recommended incubation time and temperature for ligation. Ensure that no excessive amount of wash solution remains on the sample before addition of ligation reagents. Ensure that the Ligase is active (i.e., has been kept at -20°C) and that correct dilution of the reagents have been used. Prepare fresh dilutions just before use; do not allow mix, with enzyme, to stand for more than five minutes before use.
Inefficient amplification or detection	Knowledge of the abundance of the protein target or interaction can help determine the appropriate amplification and/or detection times. The amplification and detection times can be adjusted. The recommended amplification time for highly abundant interactions is 100 minutes at 37°C. This can be extended up to overnight at 37°C for low-abundant targets or interactions. The range of detection time is 10 – 60 minutes. Detection time may need to be empirically determined. Ensure that no excessive amount of wash solution remains in the sample before addition of amplification or detection reagents. Ensure polymerase is active (i.e., has been kept at -20 °C) and that correct dilution of the reagents have been used. Prepare fresh dilutions just before use; do not allow mix, with enzyme, to stand for more than five minutes before use. Figure 5. Increased amplification time can aid in the detection of low-abundant targets.
Wash buffers used incorrectly	Wash buffer temperature can adversely affect results. Ensure Wash buffer is room temperature prior to use. Perform all washes at room temperature. Properly remove excess wash buffer prior to addition of new reagent. Residual wash buffer can cause further dilution of antibodies and/or decrease ligase and polymerase function.
Wrong filter used for acquisition	The fluorophore in the Duolink® flowPLA Detection Reagent Violet has an excitation wavelength of 390 nm, an emission wavelength of 476 nm, and may be excited using the 405 nm laser line. The fluorophore in the Duolink® flowPLA Detection Reagent Green has an excitation wavelength of 495 nm, an emission wavelength of 527 nm, and may be excited using the 488 nm laser line. The fluorophore in the Duolink® flowPLA Detection Reagent Orange has an excitation wavelength of 554 nm, an emission wavelength of 579 nm, and may be excited using the 532 nm or 561 nm laser line. The fluorophore in the Duolink® flowPLA Detection Reagent Red has an excitation wavelength of 594nm, an emission wavelength of 624 nm, and may be excited using the 532 nm or 561 nm laser line. The fluorophore in the Duolink® flowPLA Detection Reagent Far Red has an excitation wavelength of 644 nm, an emission wavelength of 669 nm, and may be excited using the 638 nm, 640 nm, or 642nm laser line. If performing multicolor flow cytometry, compensate correctly and decrease spillover from overlapping fluorescent signals. Ensure that your instrument is properly calibrated and clean before flow analysis.

Cell Retention

Probable Causes	Suggested Solutions
Cell Loss	Avoid disturbing the pellet during removal of solutions. Increase the centrifugation speed to aid in cell retention. Note: excessive centrifugation may cause clumping. Try adding BSA or FBS to the wash buffer. Use filter plates or cups. The MultiscreenHTS HV filter plate and Ultrafree MC-HV filter cups are recommended. Use cell washer instruments.
Cell Clumping	Fully resuspend cell pellet in wash buffers. Use Ca++ and M++ free buffers (addition of 1-5 mM EDTA optional). Avoid bubbles, vigorous vortexing, and excessive centrifugation. If using peripheral blood as starting sample, use an anticoagulant.

Data Acquisition

Probable Causes	Suggested Solutions
Autofluorescence	Sometimes autofluorescence is inherent to the cells or tissue sample. Try changing the detection color, e.g., from green to far red, if possible. Increase post-detection wash times or volumes with wash buffer.
Insufficient cell counts	Count cells before and after staining to determine the percentage of cell loss. Use recommended instrument specifications regarding cell concentration and flow speed for analysis. Higher cell concentrations might clog the flow cytometer and/or affect resolution. Filter the sample with cell strainer or mesh filter.
PMT voltages set incorrectly	Set the PMT voltage properly to maximize signal-to-noise ratio. No stained population should be off-scale. Keep the detectors operating in the linear range. Ensure the positive and negative are well separated.
Coalescence of Signal	If acquiring images after or during flow, extended amplification times can cause coalescence of signal. Follow recommended times for ligation and amplification steps. Too high of primary antibody titer can also cause coalescence of signal. Titrate each primary antibody using the single recognition PLA method. Figure 6. Extended amplification times can lead to coalescence of PLA signals.

Frequently Asked Questions

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