We used C57BL/6 mice at the age of 8–12 weeks where the development and maturation of RPE had been completed 59 . The mice were euthanized by CO2 inhalation and cervical dislocation. The eyes were then enucleated and bisected along the equator. The eyecups were sectioned in Ames' solution (Sigma‐Aldrich) with 10 mM HEPES and pH adjusted to 7.4, and the retina was gently removed leaving the RPE firmly attached to the eyecup. To isolate the RPE cells for patch‐clamp recordings, the eyecup was incubated at 37°C in 5% CO2 either in TrypLE Select for 15 minutes or in a solution containing (in mM) 135 TeaCl, 5 KCl, 10 HEPES, 3 EDTA‐KOH, 10 glucose, and 25 U/ml activated papain (Sigma‐Aldrich) for 30 minutes. After this, the eyecups were washed in the HEPES buffered Ames' solution supplemented with 1% bovine serum albumin (BSA; Sigma‐Aldrich). The RPE was collected by gentle trituration, stored at 37°C in 5% CO2 in the RPE culture medium and measured within 6 hours.
Activated papain
Manufactured by Merck Group
Activated papain is a proteolytic enzyme derived from the papaya fruit. It functions as a catalyst, facilitating the breakdown of proteins into smaller peptides and amino acids.
Automatically generated - may contain errors
2 protocols using activated papain
1
Isolation and Culture of Mouse RPE Cells
2
IgG Fab and Fab2 Purification
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Purified IgG (1 mg) was digested to Fab and Fc fragments by exposure to 40 μg activated papain (Sigma-Aldrich) in a solution containing 10 mM cysteine, 100 mM sodium acetate pH 5.5, 125 μM EDTA for 2–4 h at 37 °C. Fab2 was produced by digestion with pepsin (Sigma-Aldrich) for 2–4 h at 37 °C. Fab and Fab2 fragments were subsequently separated from Fc by affinity chromatography using a protein A Hi-trap column (GE Healthcare) and purified to homogeneity by size exclusion chromatography (SEC)37 (link). SDS–polyacrylamide gel electrophoresis was performed to confirm the sizes of Fab and Fab2. The NativePAGE Novex Bis-Tris gel, SimplyBlue SafeStain staining solution and protein standard were purchased from Invitrogen, Inc.
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