Figure 4.

High-Content Screening (HCS) microscopy quantifies single neurons on a full population basis. (A) Pipeline of automated HCS object identification. Green or red encircled cells indicate automatically selected or rejected objects, respectively. (B) Size-dependent identification of neuronal cell bodies. Black line delimitates 2 subpopulations according to cell size. On the left side of the threshold, the smaller cells as well as cellular debris and on the right side, the larger mature neuronal cells were identified (threshold cell body area is always = 120 µm²). (C) Size of identified neuronal cell bodies increases over the maturation time. N = 3 independent cell preparations, data were pooled from all cell culture conditions for the respective time point, >482,000 neurons/time point were included in the 2D density plots. (D) Representative HCS images of hiPSCdN cultures after 16 days, 35 days, 49 days, and 70 days of in vitro culture for cell body marker (Nissl, green), PKA-II activity (pRII, red), nociceptor marker (PKA-RIIβ, grey), and cell nuclei (DAPI, blue). hiPSCdNs, human induced pluripotent stem cell–derived nociceptors; PKA-II, protein kinase A type II. PKA-RIIβ, Protein Kinase A regulatory subunit RIIβ, DAPI, 4',6-diamidino-2-phenylindole.