Regulation of ICAM-1 in Cells of the Monocyte/Macrophage System in Microgravity
ICAM-1 surface expression reacts rapidly and reversibly to microgravity. (a) Microscopy of ICAM-1, TUNEL, HCS CellMask, and DAPI including surface calculation for HCS. In order to identify nuclei, cells were stained with DAPI (A). Apoptotic cells were identified by TUNEL reaction (B) and HCS CellMask label (C) which can be retained to a higher extend in nonapoptotic cells. ICAM-1 intensity is depicted in (D). A merge of TUNEL, DAPI, and ICAM-1 (E) shows an apoptotic cell () and a living cell . The automated calculation of an iso-surface is exclusively done for living cells using the HCS CellMask channel as shown in the merge with TUNEL, DAPI, and ICAM-1 (F). (b)–(j) BV-2 microglial cells were treated with PMA ((e), (f), and (g)) or TNF-α ((h), (i), and (j)) at the onset of microgravity or during the 1 g in-flight control phase or left untreated ((b), (c), and (d)). Cells were fixed in flight after 20 sec normogravity (1 g) (-●-) or 20 sec microgravity (μg) (-○-). Cells were stained, imaged, and analyzed as described above. The mean intensity of the ICAM-1 signal was binned into mean intensity fluorescence (MIF) categories and the number of cells (frequency) is plotted against these intensity categories ((b), (e), and (h)). ICAM-1 fluorescence intensity of all analyzed cells ((c), (f), and (i)) is depicted for normogravity (triangles) and microgravity (squares). Mean ICAM-1 fluorescence intensity of all analyzed cells ((d), (g), and (j)) was pooled for normogravity (black bar) and microgravity (open bar). For automated imaging, the unified random sampling module was utilized and 63 randomized images of each sample were recorded and at least 500 single cells from 3 independent experiments from 3 different parabolas were analyzed. Mean intensity and SEM are shown and student’s -test showed highly significant difference of the fluorescence values of , .