Technology - Applications

Reasons for performing High Content Assays on DropArray™ Cell Plates

• Assay time from fixing cells to the end of assay is reduced by at least 60%
• Number of cells required is reduced 10 times due to a 10 fold reduction in well area
• Image scan time is reduced by at least 3 times due to reduction in the number of fields that need to be scanned per well
• The cost of the assay (including reagent costs and plate costs) is reduced by at least 50%

Cellomics reagent kits and the ArrayScan HCS reader were used for the assays. We performed assays on the 96-well plate according to instructions from the manufacturer. The same procedure was followed for assays on the DropArray™ Cell Plate employing DropArray™ technology to enable rinsing of cells and to prevent assay evaporation. Activated Extracellular Signal Regulated Kinase (ERK MAPK) Assay.

This assay measures the translocation of phosphorylated ERK from the cytoplasm to the nucleus following activation of the mitogen activated protein kinase (MAPK) pathway. Phorbol 12-Myristate 13-Acetate (PMA), an activator of protein kinase C, which is upstream in the signal transduction pathway, is used to induce ERK translocation.

Figure 1: Miniaturized ERK translocation assay of NIH3T3 cells in a DropArray™ well receiving no drug treatment and in a DropArray™ well treated with 333 ng/mL PMA for 30 min.

Figure 2: ERK translocation assay performed in a 100 µL volume in a 96-well plate with primary and secondary antibodies staining time of 60 min versus a miniaturized ERK translocation assay performed in a 3 µL volume on a DropArray™ Cell Plate with primary and secondary antibodies staining time of 20 min.

Figure 3: ERK translocation assay performed in a 100 µL volume in a 96-well plate with primary and secondary antibodies staining time of 60 min to 20 min versus a miniaturized ERK translocation assay performed in a 3 µL volume on a DropArray™ Cell Plate with primary and secondary antibodies staining time from 60 min to 20 min.

Mitotic Index Assay

The mitotic index assay measures the fraction of cells undergoing cell division. The histone H3 protein becomes phosphorylated in dividing cells and this phosphorylation event is detected by immunofluorescence staining. Docetaxel, an anti-mitotic drug that binds tubulin, stabilizes microtubules and prevents their disassembly during metaphase. This gives the impression of an increased percentage of mitotic cells in cells treated with the drug.

Figure 4: Miniaturized mitotic index assay of human gastric cancer MKN7 cells in a DropArray™ well receiving no drug treatment and in a DropArray™ well treated with 1 µg/mL docetaxcel for 18 hrs.

Figure 5: Mitotic Index assay performed in a 100 µL volume in a 96-well plate with primary and secondary antibodies staining time of 60 min compared to a miniaturized mitotic index assay performed in a 3 µL volume on a DropArray™ Cell Plate with primary and secondary antibodies staining time of 20 min.

Caspase 3 Assay

Caspase 3 is an intracellular cysteine protease that is activated by cleavage at Asp175 by its upstream caspases. It plays an important role in apoptotic cell death in many different cell types. This assay measures caspase 3 activation through immunofluorescence staining of cleaved caspase 3. Doxorubicin, a chemotherapeutic agent, that causes caspase 3 activation and apoptosis, is used in this assay.

Figure 6: Miniaturized caspase 3 assay of human lung cancer A549 cells in a DropArray™ well receiving no drug treatment and in a DropArray™ well treated with 100 µg/mL doxorubicin for 5 hours.

Figure 7: Caspase 3 assay performed in a 100 µL volume in a 96-well plate with primary and secondary antibodies staining time of 60 min compared to a miniaturized caspase 3 assay performed in a 3 µL volume on a DropArray™ Cell Plate with primary and secondary antibodies staining time of 20 min.

Multiplexed Assays

The DropArray™ Cell Plate can also be used for multiplexed assays. In this example, phospho-histone H3, a marker of mitotic cells and cleaved caspase 3, a marker of apoptotic cells, are simultaneously detected by two different fluorescent antibodies.

Figure 8: Miniaturized caspase 3- mitotic index assay of human gastric cancer MKN 7 cells in a DropArray™ well receiving no drug treatment and in a DropArray™ well treated with 1 µg/mL docetaxel for 18 hours.

Figure 9: A miniaturized mitotic index- caspase 3 assay performed in a 3 µL volume on a DropArray™ Cell Plate with primary and secondary antibodies staining time of 20 min.