The culture of 5H9 hybridoma cells [49 (link)] and the production of mouse monoclonal CD9 Ab were recently described [50 (link)]. The corresponding CD9 Fab was generated using the Pierce Fab Purification kit (#44985, Thermo Fisher Scientific). Herein, CD9 Ab (500 µg) was incubated with papain immobilized on agarose resin for 3 h at 37 °C. The digested Abs were collected by centrifugation (5000× g, 1 min) using a spin column, and the flow through containing the Abs was placed in a new tube. The fragment crystalline (Fc) fragment was removed from digested Ab samples by centrifugation (1000× g, 10 min) using the NAb Protein A Plus Spin Column, and the flow through containing the purified Fab fraction was collected. The column was then washed twice with PBS and each wash fraction was combined with the Fab fraction. Using the Microsep Advance Centrifugal Device (10K molecular weight cut-off) purchased from Pall Corporation (#MCP010C46, Westborough, MA, USA), the Fab fraction was concentrated by spinning at 3000× g for 25 min at 4 °C. Concentration was then measured by absorbance at 280 nm (final yield of 0.4–0.8 mg/mL). CD9 Fab preparation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining as described in [50 (link)].
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