Duolink^® PLA Flow Cytometry Protocol

This protocol describes the use of Duolink^® PLA reagents for the detection of individual proteins, protein modifications, and protein-protein interactions within cell populations by flow cytometry.

• Materials and Equipment
• Duolink^® PLA Flow Cytometry Protocol
• Results
• Flow Cytometry Analysis
• Quick Reference Guide

Materials and Equipment

Duolink^® PLA Reagents

To run a Duolink^® PLA experiment for detection by flow cytometry, the following Duolink^® PLA products are needed:

• Duolink^® PLA Probes (one PLUS and one MINUS from different species, matching the host species of your primary antibodies). Each kit includes:
  • PLA Probes (5x) – PLUS and/or MINUS, depending on the product
  • Blocking Solution – For blocking the sample prior to antibody incubations
  • Antibody Diluent – For dilution of PLA probes, and if needed, primary antibodies

NOTE: Store all components of this kit at 4 °C. Duolink^® PLA Probemaker PLUS and/or Probemaker MINUS kits may be used to generate custom PLA probes if needed. Please see the Probemaker Guide for details.

• Duolink^® flowPLA Detection Reagent (choose from green, orange, red, and far red). Each kit includes:
  • Ligation Stock (5x) – Diluted to make 1x Ligation Buffer
  • Ligase (1 U/μL) – Added to make the Ligation Solution
  • Amplification Stock (5x) – Diluted to make 1x Amplification Buffer
  • Polymerase (10 U/μL) – Added to make the Amplification Solution
  • Detection Stock (5x) – Diluted to make 1x Detection Buffer

NOTE: Store all components at -20 °C

NOTE: The flowPLA detection kits (DUO94001-DUO94005) provide enough material to perform ~40 reactions, each with 100,000 cells in 100 µL reaction volume. Duolink PLA Probes (DUO92001—DUO92006, DUO92020 and DUO92021) will also be required.

Additional Materials needed

• Primary antibodies to detect the protein(s) of interest. Must have been raised in mouse, rabbit, or goat when used in conjunction with Duolink^® PLA Probes
• High purity water (sterile-filtered, Milli-Q^® or similar)
• Suspended cell sample, pre-treated with respect to fixation and permeabilization
• Duolink^® In Situ Wash Buffer (DUO82047)
• 1x PBS (P3813)

Equipment

• Flow cytometer with appropriate filters and software for data analysis
• 37 °C incubator
• Freezer block (for enzymes)
• Pipettes and tips (from 1 μL to 1000 μL)
• Eppendorf tubes, U-bottom or V-bottom 96-well plates
• Optional: 96-well filter plates or filter cups (Millipore UltraFree MC-HV), plate shaker

NOTE: Use the recommended centrifugation or vacuum parameters when using filter plates or filter cups.

Duolink^® PLA Flow Cytometry Protocol

The following protocol is to examine up to 100,000 cells in a 100 µL reaction volume (1,000 cells / µL). Adjust the volume as needed according to number of cells in your sample. All incubations should be performed in a 37 °C incubator. All wash steps should be performed at room temperature.

Reagent Preparation

• Duolink^® Wash Buffer and 1x PBS should be made prior to beginning the assay by dissolving the contents of one pouch in high purity water to a final volume of 1000 mL. Store 1x PBS at room temperature. Wash buffer may be stored at room temperature for short term storage (less than two weeks) or at 4 °C for long term storage.

NOTE: Bring the solutions to room temperature before use

• Many Duolink^® PLA reagents are supplied as concentrated stocks and are to be diluted immediately prior to use. Do not store diluted Duolink^® PLA reagents.

Duolink^® PLA Protocol

Before starting, suspended cell samples should be pre-treated with respect to fixation and permeabilization. Cells can be fixed, permeabilized, and blocked in bulk solution, and then aliquoted into tubes or wells for Duolink^® PLA staining unless unique conditions for these steps are being scouted.

NOTE: Duolink^® Blocking Solution and Antibody Diluent are provided with the Duolink^® PLA Probes. If alternative solutions have been optimized for primary antibody performance by traditional flow cytometry, it is likely these can be used instead.

• Blocking
  • After fixation and permeabilization, centrifuge and remove wash buffer from cells
  • Use 10 µL of Duolink^® Blocking Solution per µL volume of cell pellet (1 drop is ~30 µL)
  • Incubate in a 37 °C incubator for 60 minutes
     
• Primary Antibody Incubation
  • Dilute your primary antibody or antibodies to suitable concentration in the Duolink^® Antibody Diluent or appropriate antibody diluent
  • Aliquot 100,000 cells per well of a U-bottom or V-bottom plate.
  • Centrifuge the plate at 400 xg for 5 minutes and remove the Duolink^® Blocking Solution from the cells
  • Add the primary antibody solution to each sample and mix well
  • Incubate the plate, using the optimal incubation temperature and time for your primary antibodies
     
• Duolink^® PLA Probe Incubation
  • Dilute the PLUS and MINUS PLA probes 1:5 in the Duolink^® Antibody Diluent. For a 100 µL reaction, take 20 µL of PLA probe MINUS stock, 20 µL of PLA probe PLUS stock and 60 µL of Antibody Diluent. Make sufficient solution for all samples
  • Centrifuge at 400 xg for 5 minutes and remove the solution from the cells
  • Wash the cells twice with 200 µL of Duolink^® Wash Buffer per well, centrifuge at 400 xg for 5 minutes, and remove the solution from the cells
  • Add the PLA probe solution and mix well
  • Incubate in a 37 °C incubator for 1 hour
     
• Ligation
  NOTE: Wait to add the ligase until immediately prior to addition to the sample. Make sure ligation buffer is completely thawed and mixed well prior to usage
  • Vortex the 5x Duolink^® Ligation buffer
  • Dilute the 5x Ligation buffer 1:5 in high purity water and mix. For a 100 µL reaction, add 20 µL of the 5x Ligation buffer to 77.5 µL of high purity water. Make sufficient solution for all samples
  • Centrifuge at 400 xg for 5 minutes and remove the solution from the cells
  • Wash the cells twice with 200 µL of Duolink^® Wash Buffer per well, centrifuge at 400 xg for 5 minutes, and remove the solution from the cells
  • During the wash, retrieve the Ligase from the freezer using a freezer block (-20 °C)
  • Add Ligase to the 1x Ligation buffer at a 1:40 dilution and mix. For 100 µL ligation solution, add 2.5 µL of Ligase to 97.5 µL of the 1x ligation buffer
  • Add the ligation solution and mix well
  • Incubate in a 37 °C incubator for 30 minutes

• Amplification
  NOTE: Wait to add the polymerase until immediately prior to addition to the sample.
  • Vortex the 5x Duolink^® Amplification buffer
  • Dilute the 5x Amplification buffer 1:5 in high purity water and mix. For 100 µL reaction, add 20 µL of the 5x Amplification buffer to 78.75 µL of high purity water. Make sufficient solution for all samples
  • Centrifuge at 400 xg for 5 minutes and remove the solution from the cells
  • Wash the cells twice with 200 µL of Duolink^® Wash Buffer per well, centrifuge at 400 xg for 5 minutes, and remove the solution from the cells
  • During the wash, retrieve the Polymerase from the freezer using a freezer block (-20 °C)
  • Add Polymerase to the 1x Amplification buffer at a 1:80 dilution and mix. For 100 µL amplification solution, add 1.25 µL of Polymerase to 98.75 µL of the 1x amplification buffer
  • Add the amplification solution and mix well
  • Incubate in a 37 °C incubator for 100 minutes

  NOTE: Longer amplification time (up to overnight) may be required for low abundance proteins or protein interactions

• Detection
  NOTE: The Duolink^® Detection Buffer is light-sensitive. Protect from light.
  • Vortex the 5x Detection Buffer
  • Dilute 1:5 in high purity water and mix. For 100 µL reaction, add 20 µL of the 5x Detection buffer to 80 µL of high purity water. Make sufficient solution for all samples
  • Centrifuge at 400 xg for 5 minutes and remove the solution from the cells
  • Wash the cells twice with 200 µL of Duolink^® Wash Buffer per well, centrifuge at 400 xg for 5 minutes, and remove the solution from the cells
  • Add the detection solution and mix well
  • Incubate in a 37 °C incubator for 30 minutes

  NOTE: Detection times can be adjusted (from 10-60 minutes) depending on protein abundance or level of background.

• Final Washes
  • Centrifuge at 400 xg for 5 minutes and remove the solution from the cells
  • Wash the cells with 200 µL of Duolink^® Wash Buffer per well, centrifuge at 400 xg for 5 minutes, and remove the solution from the cells
  • Resuspend the cells in 1x PBS at ~1,000 cells / µL

Results

Flow Cytometry Settings

Perform flow cytometry analysis using appropriate instrument settings. It is recommended to optimize the instrument settings for each individual cell line on fixed, unstained cells (i.e., without the Duolink^® PLA steps) prior to running the experiment. Once this has been determined, the same settings should be used for all the samples within the experiment. Use forward and side scatter parameters to gate on fixed cells and to remove cellular debris. Optionally, doublets can be gated out by using side scatter width or area vs. height parameters.

Technical negative controls should include omission of each primary antibody separately and omission of all primary antibodies. These controls will determine non-specific binding of each primary antibody and the Duolink^® PLA probes, respectively. It is important to note that the negative controls (e.g., no primary antibodies but with PLA probes) may have increased background fluorescence when compared to fixed, unstained cells. The technical negative control (e.g. no primary antibodies but with PLA probes) can be utilized to set the gate for the baseline Duolink^® PLA fluorescence  measurement and applied to all experimental samples

Flow Cytometry Analysis

Figure 1. Increased amplification time during the Duolink^® PLA experiment can aid in the detection of low-abundant protein targets by flow cytometry. Duolink^® PLA technology 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 low-abundant protein events, such as EZH2-H3K27me3 interactions, by conventional flow cytometry. C) Combining Duolink^® PLA with imaging flow cytometry allows localization of proteins or protein events (interactions or modifications) in large cell populations.

Quick Reference Guide

Cell samples must be properly fixed and permeabilized as suitable for the primary antibodies of choice before proceeding. The following protocol is to examine up to 100,000 cells in a 100 µL reaction volume (1,000 cells / µL). Adjust the volume as needed for the number of cells in your sample. Keep cells in suspension. Avoid clumping.

Step	Instructions
Blocking	Add Blocking Solution and incubate for 1 hr at 37 °C Centrifuge and remove blocking solution
Primary Antibody Incubation	Dilute primary antibodies to suitable concentration in Antibody Diluent Incubate as appropriate for optimal antibody performance Wash twice in 1x Duolink PLA Wash Buffer
PLA Probe Incubation	Dilute PLA probes 1:5 in Antibody Diluent (PLUS and MINUS) Add diluted PLA Probes and incubate for 1 hr at 37 °C Wash twice in 1x Duolink PLA Wash Buffer
Ligation	Make 1x ligation buffer by diluting 5x Ligation buffer 1:5 in high purity water and mix Dilute ligase enzyme 1:40 in 1x Ligation buffer Add ligation solution and incubate for 30 min at 37 °C Wash twice in 1x Duolink PLA Wash Buffer
Amplification	Make 1x amplification buffer by diluting 5x Amplification buffer 1:5 in high purity water and mix Dilute ligase enzyme 1:40 in 1x Ligation buffer Add amplification solution and incubate for 100 min* at 37 °C Wash twice in 1x Duolink PLA Wash Buffer
Detection	Make 1x detection buffer by diluting 5x Detection buffer 1:5 in high purity water and mix Add detection solution and incubate for 30 min* at 37 °C Wash twice in 1x Duolink PLA Wash Buffer Resuspend cell sample in 1x PBS at 1000 cells/mL for analysis
*Incubation conditions may vary depending on abundance of protein

References

1. Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius K, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson L, et al. 2006. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods. 3(12):995-1000. https://doi.org/10.1038/nmeth947

2. Leuchowius K, Weibrecht I, Landegren U, Gedda L, Söderberg O. 2009. Flow cytometricin situproximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family. Cytometry. 75A(10):833-839. https://doi.org/10.1002/cyto.a.20771

3. Avin A, Levy M, Porat Z, Abramson J. 2017. Quantitative analysis of protein-protein interactions and post-translational modifications in rare immune populations. Nat Commun. 8(1): https://doi.org/10.1038/s41467-017-01808-6

4. Andersen SS, Hvid M, Pedersen FS, Deleuran B. 2013. Proximity ligation assay combined with flow cytometry is a powerful tool for the detection of cytokine receptor dimerization. Cytokine. 64(1):54-57. https://doi.org/10.1016/j.cyto.2013.04.026