TB staining of the ERM may minimize mechanical trauma to the retina during ERM removal and should allow the recognition of the whole extent of the ERM, which has led to good surgical outcomes and minimization of the recurrence of ERM in several clinical studies. TB exhibits outstanding affinity for ERM because of the strong presence of dead glial cells within those membranes. Currently, state-of-the-art TB usage recommends blue dye application mainly for ERM staining. Although the use of TB for ILM staining may also be feasible, in our experience ILM visualization is much more difficult than with the ICG- or BriB-guided procedure. TB has been proposed to stain preretinal tissues such as ILM and ERM in chromovitrectomy ( Figure 48.2). Eduardo Büchele Rodrigues MD, in Retinal Pharmacotherapy, 2010 TRYPAN BLUE The concentration of the trypan blue solution used is typically 0.4% (w/w). Thus, when Trypan blue is utilized, the fluorescence detected by flow cytometry is from the cells with internalized fluorescent MSN or cell autofluorescence. Trypan blue cannot penetrate the membrane of live cells, it will quench all the fluorescence from the physically adsorbed MSN on cell membrane. Only live cells that have internalized the FITC-MSN report a positive result. By adding trypan blue to the solution, the extracellular FITC-MSN material is quenched, excluding them from the quantification. When these two events occur, it is often difficult to distinguish between them by flow cytometry. Fluorescein labeled MSN can be either internalized by cells or they can physically adsorb to the exterior of the cell membrane. Trypan blue is also used to eliminate false positives from occurring during cell counting by flow cytometry ( Radu et al., 2004 Slowing et al., 2006 Trewyn et al., 2008). Under light microscopy analysis, only dead cells have a blue color. In a dead cell, trypan blue passes through the porous cell membrane and enters the cytoplasm. Because live cells have an intact cell membrane, trypan blue cannot penetrate the cell membrane of live cells and enter the cytoplasm.
Trypan blue is a stain used to quantify live cells by labeling dead cells exclusively. Trewyn, in Methods in Enzymology, 2012 2.6.3 The purpose of the trypan blue