Multiplexed Measurement of 64 Doses
In order to assess the suitability of the ImageStream for high-content screening, we applied the method of Krutzik & Nolan to multiplex 64 THP1 cell samples, each having a different TNF-α dose and exposure time.
After treatment, the individual samples were stained with three different Alexa Fluor succinimidyl ester dyes (AF405, 488, 660), each at one of four intensities, to yield 64 unique dye/intensity combinations. The samples were then mixed, labeled with a rabbit anti-NF-kB polyclonal antibody, a Cy3-anti-rabbit secondary antibody, and propidium iodide, before being run on the ImageStream. Six doses at five time points were tested in duplicate, plus two negative control samples. Imagery of >100,000 cells (about 600,000 images) was acquired in approximately 10 minutes.
Figure 2 demonstrates the ability to resolve the multiplexed cell samples. The intensity histogram resolves four comingled cell populations labeled with Alexa Fluor 660, and the AF405 versus AF488 intensity scatter plot demonstrates the ability to resolve 16 samples. Extending this to three dimensions with four intensities for three dyes results in resolution of 64 samples per well.
Figure 3 includes a heat map table of the mean Similarity scores calculated from each of the multiplexed samples and organized by dose (vertically) and time point (horizontally).
Each cell of the table is also color coded by mean Similarity score, with high Similarity scores shown in red and low scores shown in green. As expected, the highest Similarity scores corresponded to the highest doses and latest time points. Figure 3 also shows example imagery from two of the multiplexed samples.
The mean Similarity values were also plotted as dose-response curves (Figure 4), with each curve generated using a different TNF-α exposure time. Doses above 100 pg/mL and exposure times longer than 15 minutes caused significant NF-kB translocation.
The ImageStream platform can perform 64X multiplexing of NF-kB translocation assays in nonadherent cells through the use of intensity coding. The recent availability of an autosampler for the ImageStream system now allows the independent measurement of translocation within 64 samples per well, for up to 6,144 samples per 96-well plate.