From: In Vitro Interactions between Peripheral Blood Lymphocytes and the Wong-Kilbourne Derivative of Chang Conjunctival Cells Invest. Ophthalmol. Vis. Sci.. 2012;53(3):1492-1498. doi:10.1167/iovs.11-7708 Figure Legend: (A–C) Cell morphology and interaction analyses in cocultures with cell membrane staining. Green: epithelial cells with PKH67; red: lymphocytes with PKH26. (A) Micrographs show the same area with a green (Aa) or a red (Ab) filter with fluorescence microscopy. WKD cells were cocultured with PBLs in direct contact for 24 hours. Plasmic membrane exchanges between the two cell types were observed after 1 day of coculture, only if the epithelial cells were in direct contact with the PBLs (WKD cells appeared in both green and red, meaning that a stain transfer between the two cell types may have occurred). Controls (data not shown)—either WKD cells alone or WKD cells cocultured with PBLs separated by a 0.4-μm pore translucent PET membrane—showed exclusively green WKD cells, without any transfer of red stain by the PBLs to the WKD cells. (B) Micrographs show the same area using either a green (Ba) or a red (Bb) filter, by confocal microscopy. WKD cells were cocultured with PBLs separated by a 0.4-μm pore translucent PET membrane for 24 hours. WKD cells appeared in green only, meaning that without any direct cell contact, a stain transfer between the two cell types may not have occurred. These controls showed exclusively green WKD cells, without any transfer of red stain by the PBLs to WKD cells. (C) Micrographs show the same area (Ca , Cc) using either a green filter (Ca, Cb) or a red (Cc, Cd) filter by confocal microscopy. (Cb, Cd) The same area in a vertical section (two-headed arrow). WKD cells were cocultured with PBLs in direct contact for 90 minutes. Plasmic membrane exchanges between the two cell types were observed after 90 minutes of coculture, only if the epithelial cells were in direct contact with the PBLs (WKD cells appeared in both green and red, indicating that a staining transfer between the two cell types may have occurred). Yellow staining was due to overexposure during fluorescence microscopy. (D) Cell morphology and interaction analyses in coculture using SEM. Note that tight connections between PBLs (short arrow) and WKD cells (long arrows) appeared like cell membrane fusion. (E) Cell morphology analysis in coculture using cell membrane standard immunofluorescence staining on WKD and PBL cells. Cytoskeleton (F-actin) was stained in green by phalloidin. Phalloidin staining showed the morphology of both cell types after 24 hours of coculture. Note that PBLs showed cytoplasmic expansions (arrows), in contact with WKD cells. Date of download: 11/6/2017 The Association for Research in Vision and Ophthalmology Copyright © 2017. All rights reserved.