For phagocytosis assay confluent RPE cells on collagen I coated cover slips were used (please refer to Section 4.5 ). After treatment, phagocytosis staining was conducted as previously established [1 (link)]. Fluorescence latex beads (Sigma-Aldrich) were opsonized with photoreceptor outer segments prepared from porcine retinae. Opsonized beads were applied to the RPE cells and incubated for four hours. Cells were washed with media and PBS to remove excess beads. Cells were fixated with paraformaldehyde and mounted with Fluoromount-G™ with DAPI (Thermo Fisher Scientific). Fluorescence images were taken with Axiovert Imager M.2 and AxioVision Software (both Zeiss, Jena, Germany). The DAPI stained cell nuclei were counted per hand. Fluorescence beads were counted by self-programmed macro in Fiji (ImageJ2, https://imagej.net/software/fiji/downloads (accessed on 8 August 2022). For evaluation measured beads were set in relation to number of cell nuclei.
Axiovert imager m 2
Manufactured by Zeiss
Sourced in Germany
The Axiovert Imager M.2 is a high-performance laboratory microscope designed for image acquisition and analysis. It features a motorized stage, autofocus, and integrated camera, enabling precise and reproducible image capture. The microscope is capable of a variety of imaging techniques, including brightfield, phase contrast, and fluorescence. Detailed specifications and performance characteristics are available upon request.
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2 protocols using axiovert imager m 2
1
Phagocytosis Assay for Retinal Pigment Epithelial Cells
2
Phagocytosis Assay in RPE Cells
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Confluent RPE cells seeded on collagen I coated cover slip were treated with 50 μg/mL FV fucoidan and inflammatory agents LPS, Poly I:C, TNFα and Pam, respectively, for one, three and seven days and an established phagocytosis assay was conducted [63 (link)]. In brief, fluorescent labeled latex beads (Sigma-Aldrich; #L1030) and photoreceptor outer segments (POS) from porcine retinae were mixed and given to the cells 4 h after treating them with the respective stimuli. After fixating and washing steps, the cells were mounted with Fluoromount-G™ and DAPI (Thermo Fisher Scientific; #00-4959-52). Fluorescence microscope Axiovert Imager M.2 and AxioVision Software (both from Zeiss Microscopy GmbH, Germany) were used for image capture. Cell nuclei, stained with DAPI, were counted manually. For counting of fluorescent beads, a self-designed macro from Fiji was used (https://imagej.net/software/fiji/downloads ). Uptake of beads was set in relation to cell nuclei number for evaluation.
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