Zen2011 blue

Analysis of cells was completed using an Olympus BX54WI Fluorescent microscope with a Carl Zeiss AxioCam (Zen 2.3 Blue edition, Carl Zeiss Microscopy GmbH, White Plains, NY, USA) and was used to image all cell populations by a trained cytologist. A Zen2011 Blue 3.0 (Carl Zeiss) was used to process the images and further analyze the cells. CAMLs and CTCs are detected in this process by their phenotypic expression of CD45, Cytokeratins 8, 18, 19, and DAPI, as previously described [19 (link),26 (link)] (Figure 1). CAMLs were identified by their enlarged polynucleated nucleus (ranging from 14 to 64 microns) and by their giant cellular bodies (roughly 30 to 300 microns in length.) Other tumor-associated cells such as circulating tumor cells (CTCs) and epithelial-to-mesenchymal transition cells (EMTs) can be identified through these filtration methods, however, they were not included in the analysis of MN, as MN were not found in these cell populations. After identification of the cell populations, DAPI positive events were identified by the Zen2011 Blue (Carl Zeiss) and presented to a cytotechnician. MN were defined as small circular DAPI positive structures found within the cytoplasmic region of each cell, but distinct and separate from the primary nucleus.

Data were analysed using the Zeiss ZEN 2011 (Blue) software package. Diverse measurements were taken including the 2-dimensional area (μm²) of each ovule tissue of interest, using the “contour (spline)” graphics tool to encircle the tissue, as well as the longitudinal and transverse dimensions (μm) of the same tissues, using the “line” graphics tool, and the antipodal nuclei were counted using the “event marker” graphics tool (Fig. 4c). Measurements were taken by following tissue boundaries for each given trait throughout optical sections and placing contour markers at the widest point. Two-dimensional ovule area was measured at the boundary between integument and nucellus. Embryo sac area was measured by tracing the outline of the structure from the micropyle to the chalazal region. The residual somatic cell (nucellus) area was measured by subtracting the embryo sac area from the whole ovule area.

Samples are filtered with a CellSieve™ Microfiltration Assay using a low-pressure vacuum system which isolates CTCs based on size exclusion, >7 micron, as previously described1 (link)2 (link)3 (link)25 (link)39 . Cells were stained and identified by fluorescent enumeration using CTC enumeration stains, as previously described. Briefly, a low-pressure system uses a filter holder assembly with CellSieve™ filter attached. Peripheral blood (7.5 mL), is diluted in a prefixation buffer and drawn through the filter. The filter is washed, postfixed and permeabilized. The captured cells are stained with an antibody cocktail consisting of FITC-anti-Cytokeratin 8, 18, 19, PE-anti-EpCAM and Cy5-anti-CD45 for 1 hour and mounted with Fluoromount-G/DAPI (Southern Biotech). An Olympus BX54WI Fluorescent microscope with Carl Zeiss AxioCam was used to image the samples. Exposures were preset as 2 sec (Cyanine5), 2 sec (PE), 100–750 msec (FITC), and 10–50 msec (DAPI) for equal signal comparisons between cells. A Zen2011 Blue (Carl Zeiss) with AxioVision Mark and Find module was used to process the images, mark the x/y placement of the cells, and relocate previously imaged cells in a semi-automated manner. Samples were archived and placed in storage at 4 °C for 1 week-2 years.