1. Store all media at 4°C and use within 4 weeks.
2. To make up the acid, goggles must be worn because of the exothermic nature of the reaction.
3. Maintaining the epithelial surface on the side of the black clip prevents confusion in later experiments.
4. Cryopreservation of amnion was not used (23) since freezing the amnion may produce breaks in otherwise healthy tissue that would then affect further studies, particularly the permeability studies.
5. For all RPE assays, it is recommended that the cells be used prior to passage 19 in order to maintain integrity of the phe-notype. The cells should be regularly checked for phenotype by using antibodies to CRALBP.
6. For HUVEC assays, cells are used from passages 2 to 3 and are routinely checked for their endothelial nature by using antibodies to vascular specific VE-cadherin.
7. The authors prefer the use of neutralisation of the native epithelium of the amnion rather than using Thermolysin.
8. The flow system can be used to measure permeability of the trilayer to known tracers (see below).The flow rates for upper and lower chamber can be altered as necessary but if used at 0.1 mL/min, a shear stress of less than 0.25 dyne/cm2 is present. This produces negligible shear stress for cells (17).
9. All steps are 100 mL per well. Wash with PBS containing 0.01% Tween between each step. Use 1:250 dilution (44 mL in 11 mL).
10. The reason for flow being present is also to ensure absence of any unstirred boundary layers, which would lead to an underestimation of solute flux.
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