Figure 1.

A wing dissociation protocol to release cells from cuticle and minimize cell death and loss. (a) Workflow diagram of assays compatible with gentle dissociation on larval and pupal tissues. Pupal tissues after 6 h into metamorphosis are encased within pupa cuticle and require dissociation for the subsequent assays. (b) Diagrams of pupal wing morphogenesis during metamorphosis. Notum (pink) is present in larval wings but absent from dissected pupa wings after 18 h APF. Larval and pupal wings contain hinge (yellow) and wing pouch (blue). (c) Diagram of pupa dissection (dotted lines) with image of 24 h APF pupa removed from the tanned pupal case. Wings are enclosed in shiny, translucent pupa cuticle and are manually dissected from the body at the hinge for dissociation. (d) Example flow cytometry plot of dissociated 24 h APF pupal wings. Cells were stained with a vital DNA dye (DyeCycle Violet) to discern cells from debris. Cell viability was assayed using a propidium iodide (PI) permeability assay and dead or dying cells were quantified based on gating of PI-positive cells. (e) Quantifications of viable vs. dead/dying cells in trypsin-based dissociation vs. collagenase/dispase dissociation in 24 h APF pupal wings and 24 h APF pupal eye/brain complexes.