Paul.xu at 22:01, 2 January 2018

2018-01-02T22:01:42Z

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== HER2 ==

Anti-HER2 hybridomas were produced, as described previously.(15) In brief, BALB/c mice were immunized using intraperitoneal (i.p.) injections of 100 mg of recombinant HER2-extracellular domain together with Imject Alum (Thermo Fisher Scientific, Inc.). After several additional immunizations, a booster injection was intraperitoneally administered 2 days before harvesting spleen cells. Spleen cells were then fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). The resulting hybridomas were grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.). Culture supernatants were screened using enzyme-linked immunosorbent assay (ELISA) with recombinant HER2-extracellular domain. MAbs were purified from the supernatants of hybridomas, cultured in Hybridoma-SFM (Thermo Fisher Scientific, Inc.) using Protein G Sepharose 4 Fast Flow (GE Healthcare UK Ltd.).

== CD133 ==

Cell lines LN229, HCT-116, Chinese hamster ovary (CHO)-K1, Caco-2, and P3X63Ag8U.1 (P3U1) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). LN229/CD133 and CHO/CD133 were produced by transfecting pCAG/PA-CD133-RAP-MAP into LN229 and CHO-K1 cells using Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA) and Lipofectamine LTX (Thermo Fisher Scientific, Inc.), respectively. A few days after transfection, PA tag-positive cells(16) were sorted using a cell sorter (SH800; Sony Corp., Tokyo, Japan). CHOK1, CHO/CD133, and P3U1 cell lines were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and LN229, LN229/CD133, HCT-116, and Caco-2 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 units/mL of penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified atmosphere containing 5% CO2 and 95% air.

Production of hybridoma

Female 4-week-old BALB/c mice were purchased from CLEA Japan (Tokyo, Japan). Animals were housed under specific pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved all of the animal experiments described herein. Mice were immunized using intraperitoneal (i.p.) injections of LN229/CD133 cells together with Imject Alum (Thermo Fisher Scientific, Inc.). After several additional immunizations, a booster injection of LN229/CD133 cells was intraperitoneally administered 2 days before harvesting spleen cells. Spleen cells were then fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN) or GenomONE-CF (Ishihara Sangyo Kaisha, Ltd., Osaka, Japan). The resulting hybridomas were grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.). Culture supernatants were screened using flow cytometry (first screening), Western blot (second screening), and immunohistochemical analyses (third screening). MAbs were purified from supernatants of hybridomas cultured in Hybridoma-SFM medium (Thermo Fisher Scientific, Inc.) using Protein G Sepharose 4 Fast Flow (GE Healthcare United Kingdom, Ltd., Buckinghamshire, England). Flow cytometry Cells were harvested by brief exposure to 0.25% trypsin/ 1 mM ethylenediaminetetraacetic acid (EDTA) (Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin (BSA)/phosphate buffered saline (PBS), the cells were treated with 1 mg/mL of anti-CD133 mAb (clone CMab-43) for 30 min at 4C and then with Alexa Fluor 488-conjugated antimouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA). Fluorescence data were collected using EC800 or SA3800 Cell Analyzers (Sony Corp.). 
[[Category:Antibodies]]

Monoclonal Antibody Methods - Revision history

Paul.xu at 22:01, 2 January 2018