Discovering Molecules That Regulate Efferocytosis Using Primary Human Macrophages and High Content Imaging
Fig 7
Monoclonal antibodies capable of modulating efferocytosis are identified using high content image-based screening.
Purified hybridoma supernatants were tested in a 384-well format using day 7 MΦ’s co-cultured with apoptotic Jurkat cells then analyzed on the Operetta High Content Imager. Results are expressed as PI (z-axis) and % positive cells (y-axis) for each sample (A) with controls represented as follows: cytochalasin D treated MΦ’s with apoptotic cells negative control (green squares); live cell, baseline phagocytosis (dark red squares); apoptotic cells with no antibody (blue squares); dexamethasone treated MΦ’s with apoptotic cells positive control (orange squares). Samples from each plate are grouped and represented by colored squares. The antibody panel contains molecules that exhibit positive and negative effects on MΦ efferocytosis as revealed by comparison of PI of treated cells to controls (B). Each diamond represents the PI data from an individual hybridoma and each is compared to the PI of MΦ’s incubated with apoptotic cells in absence of antibody (red line) which is geometric mean of 4 replicates 9.995 ± 7.54. Data shown are from testing antibodies on macrophages from a single donor, a second assay using cells from a different donor was also performed yielding similar results.