Figure 4. Expression of CD40 on various subsets of retinal cells. Single-cell suspensions were stained with isotype control or with anti-CD40 mAb. Cells were coincubated with antibodies that detect cell surface expression of CD11b, CD31, CD105, or Thy-1 (A), or cells were permeabilized followed by incubation with antibodies against rhodopsin and vimentin (B), or GFAP (C) to detect intracellular expression of these markers. PE-conjugated anti-CD40 mAb was used in all conditions except when staining GFAP⁺ cells, where FITC-conjugated mAb was used instead. The average expression of CD40 (cMFI) on gated CD11b⁺, CD31⁺, CD105⁺, Thy-1⁺, GFAP⁺, rhodopsin⁺, and vimentin⁺ events were calculated as described in Methods. Data are shown as the mean±SEM from three to four independent experiments. CD40 was not detected on rhodopsin⁺ and GFAP⁺ cells. D: Representative dot plots of CD40 versus various markers are shown. Red dots represent cells that expressed these markers. Horizontal and vertical lines represent gates obtained with isotype control antibodies.