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. 2023 Sep 14;14:1167965. doi: 10.3389/fimmu.2023.1167965

Figure 8.

Figure 8

GPI-scFv X5 is transferred from CEMss cells to dendritic cells. GPI-scFv X5 CEMss cells were cocultured with DiD dyed DCs for 2 hours and analyzed by Amnis ImageStream Mark-II. (A) Gating strategy for analysis of DC : TC cocultures. RMS feature of Ch1 (Bright-field) were used to plot gradient by normal frequency to select for focused events. Cells were then analyzed by aspect ratio vs area to identify single cells and likely doublets. Cells were finally selected for positivity in both Ch02 and Ch11 looking for expression of eGFP and DiD, respectively. (B) Population count of total cells sorted and counts of expression for GFP positive, DiD positive, or double positive (DP) populations and relative percent. (C) Representative images of CEMss GPI-scFv X5 co-expressing GFP and GPI-HA-scFv X5 (top panel), DC : TC doublets show anti-HA PacBlue tagged GPI-HA-scFv X5 detected on DCs after DC : TC coculture (middle panel), and DiD dyed DC positive for GPI-HA-scFv X5 (bottom panel). (D) Coexpression of HA PacBlue and GFP on GPI-scFv X5 CEMss cells are 94.4% double positive. (E) DiD dyed DCs analyzed for coexpression of DiD and HA PacBlue after 2 hours of coculture with GPI-HA-scFv X5 CEMss reveals two distinct population of DiD+ HA PacBlue+ (48.9%) and DiD+ HA PacBlue- (42.3%).