Sa3800 cell analyzer

HSC-3, MKN45, NUGC-4, CHO-K1, CHO/CD44s, CHO/CD44v3–10, and CHO/CD44v3–10 deletion mutants were obtained using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). In the CBIS screening and epitope mapping, the hybridoma supernatants were treated with CHO-K1, CHO/CD44v3–10, or CHO/CD44v3–10 deletion mutants. In the dose-dependent assay, the cells were incubated with C₄₄Mab-94, C₄₄Mab-46, or control blocking buffer (0.1% BSA in PBS). Next, the cells were treated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA). The data were analyzed using the EC800 Cell Analyzer and EC800 software ver. 1.3.6 (Sony Corp.), or the SA3800 Cell Analyzer and SA3800 software ver. 2.05 (Sony Corp.).

Flow cytometry was performed as previously described^{8 (link)}. Fluorescence data were collected using a FACSCanto II system (BD Biosciences), a SA3800 cell analyzer (Sony Biotechnology, Tokyo, Japan), or a MA900 cell sorter (Sony Biotechnology) according to staining using an anti-CD271 antibody (ME20.4; BioLegend, San Diego, CA, USA). Data were analyzed using FlowJo software (v10, FlowJo LLC, Ashland, OR, USA) and the CytoExploreR package in R software²¹ .

The cells were harvested following a brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells were washed with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and treated with primary mAbs for 30 min at 4 °C. Thereafter, the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA) or Oregon Green anti-rat IgG (1:2000; Thermo Fisher Scientific Inc.). Then, fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).

The cells were harvested following brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.), washed with 0.1% bovine serum albumin (BSA)/phosphate-buffered saline (PBS), and treated with primary mAbs for 30 min at 4 °C. Thereafter, the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA). Fluorescence data were collected using a SA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).

CHO/CD44v3–10, CHO-K1, and HSC-3 were harvested using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells were treated with C₄₄Mab-34, C₄₄Mab-46, or blocking buffer (control) (0.1% BSA in PBS) for 30 min at 4 °C. Then, the cells were treated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:2000; Cell Signaling Technology, Inc, Danvers, MA, USA) for 30 min at 4 °C. The SA3800 Cell Analyzer and SA3800 software ver. 2.05 (Sony Corporation) were used for fluorescence data collection and analysis, respectively.

CHO/CD44v3–10 and CHO-K1 cells were prepared using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). COLO201 and COLO205 were obtained by pipetting. The cells (1 × 10⁵ cells/sample) were incubated with C₄₄Mab-1, C₄₄Mab-46, or blocking buffer (0.1% BSA in PBS; control) for 30 min at 4 °C. Then, the cells were treated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:2000; Cell Signaling Technology, Inc.) for 30 min at 4 °C. Fluorescence data were collected and analyzed using the SA3800 Cell Analyzer and SA3800 software (ver. 2.05, Sony Corp.), respectively.

The 6-week-old female BALB/c mice were purchased from CLEA Japan (Tokyo, Japan). Mice were housed under specific pathogen-free conditions. To minimize animal suffering and distress in the laboratory, all mice experiments were performed according to relevant guidelines and regulations. Our animal experiments were approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001). Mice were monitored every day for health during the period of experiments. Mice were intraperitoneally immunized with CHO/CD44v3–10 (1 × 10⁸ cells) with Imject Alum (Thermo Fisher Scientific Inc.) as an adjuvant. We performed additional immunizations of CHO/CD44v3–10 (1 × 10⁸ cells, three times) and performed a booster injection of CHO/CD44v3–10 (1 × 10⁸ cells) 2 days before harvesting the spleen cells. We used polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN, USA) to fuse the splenocytes and P3U1 cells. The hybridoma supernatants, which are negative for CHO-K1 cells and positive for CHO/CD44v3–10 cells, were selected using SA3800 Cell Analyzer (Sony Corp. Tokyo, Japan).