Proximity Ligation Assay (PLA): HTS & Microtiter Plate Guidelines

The Duolink In Situ PLA is a robust assay, and can be developed for high-throughput screens (HTS). However, we recommend you first optimize the assay in chambered slides, especially focusing on defining the appropriate antibody concentrations. Once you have established the experimental parameters for your antibodies, then you can scale-up to microtiter plates in a stepwise manner; first, to a single plate and then on to planning for the logistics associated with larger numbers. For general considerations regarding the use of cells in performing PLA, which are equally applicable here, please refer to the Protocols section of our website and the document entitled "Cell Culture Guidelines".

1. Choice of cell line is especially important when developing PLA for HTS. For the microtiter plate format, we recommend cell lines that grow adherently. Cells grown in suspension are simply not amenable to this format due to the many wash steps associated with the assay.
2. The use of ELISA-style plate washing systems is not recommended for high-throughput PLA. These systems do not have adequate control over the liquid dispensing pressures, such that they will typically remove most of the cells from your plates. If more advanced robotic liquid handling systems are unavailable, then we recommend doing liquid transfers with a multichannel micropipette. Although this will restrict the number of plates that can be processed in each experiment, you will obtain far superior results and have fewer repeat experiments to perform.
3. As when performing the assay on slides, it is important not to allow your samples to dry in between the different steps, as this can greatly increase non-specific fluorescence. Generally, when robotic systems are being employed for liquid handling, then this is a less important consideration. However, if liquid transfers are done using a multichannel micropipette, then this becomes much more important and should be addressed as part of assay development.
4. Choice of 96well or 384well format will depend on the availability & capability of image-capture and robotic/automated liquid handling systems. We recommend restricting the assay format to 96well for several reasons, mostly related to problems with liquid handling. Researchers often experience significant problems with bubble-formation when using the 384well format due to the repeated steps involving wash solutions. However, if good robotic platforms are available for plate processing, and the imaging system can accommodate them, then the 384well format is possible.
5. Choice of microtiter plates is critical for both good imaging and avoiding problems with automated liquid handling systems. Please refer to the guidelines published by the manufacturer of your imaging system as well as any robotic system to ensure conformity with both plate dimensions and optics. We recommend avoiding the use of lift-arms for moving plates. Moving them by hand both on and off the stage/platform of a robotic system will help reduce imaging problems due to scratched plates.
6. Reaction volumes for the different incubation steps can be optimized based on your experimental set-up and plate choice, but care needs to be taken to avoid drastic volume changes which may negatively affect the enzyme-containing assay steps. Potential steps for volume optimization includes primary antibody, secondary antibody (for secondary-conjugate form of the assay), ligation, and amplification. As a starting point, we recommend using 40uL reaction volumes for 96well plates, and 20uL reaction volumes for 384well plates. Washing steps should be done using 200uL volumes for 96well plates, and 75uL volumes for 384well plates.
7. We recommend performing the primary antibody incubation over-night for all HTS formats, regardless of whether the assay being performed is a primary- or secondary-conjugate form. For most antibody pairs, this extended primary incubation can be done at room temperature, and the over-night scheduling will usually provide a better work flow for performing the assay.
8. The reactions involving the ligation & amplification steps are especially critical to obtaining good results. Prior to applying these solutions it is important to try to remove as much residual liquid as possible from the wells of your plates, but to do so without allowing them to fully dry. Residual liquids will affect the buffering conditions of these enzyme-containing solutions, reducing their effectiveness. A good method of addressing these conflicting needs is to invert the plates after the last wash step, and tap them gently on a paper towel before adding the reaction solution.
9. For several steps of the assay, we recommend that plate processing be done in stages to avoid large differences in timing between the first and last plates of the experiment. This is especially critical for the following steps: length of time for secondary antibody incubation (for secondary-conjugate form of the assay), length of time for the ligation incubation, length of time for the amplification incubation.
10. Attention should be paid to avoid variations in temperature associated with how the microtiter plates are situated during the 37degree incubations for the ligation and amplification steps of the assay. Variations in temperature can greatly affect the quality of your results, especially if there is well-to-well variability that could affect your final data analysis. We recommend that plates either be placed individually, or stacked in pairs, directly on the shelves within the 37degree incubator. Various commercial heat-transfer blocks are available for reducing temperature variability, but these are not practical when assays involve more than a few plates.

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