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.
To run a Duolink® PLA experiment for detection by flow cytometry, the following Duolink® PLA products are needed:
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.
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.
NOTE: Use the recommended centrifugation or vacuum parameters when using filter plates or filter cups.
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.
NOTE: Bring the solutions to room temperature before use
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.
NOTE: Longer amplification time (up to overnight) may be required for low abundance proteins or protein interactions
NOTE: Detection times can be adjusted (from 10-60 minutes) depending on protein abundance or level of background.
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
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.
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.