All media were supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc.), 100 units/mL of penicillin, 100?g/mL of streptomycin, and 25?g/mL of amphotericin B (Nacalai Tesque, Inc.). cell line sensitively in flow cytometry. ? PMab-219 is useful for IHC using paraffin-embedded cell sections. 1.?Introduction Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in normal tissues including renal podocytes, type I lung alveolar cells, and lymphatic endothelial cells [1,2]. The interaction between PDPN on lymphatic endothelial cells and C-type lectin-like receptor-2 (CLEC-2) on platelets has been shown to facilitate embryonic blood/lymphatic vessel separation [1,[3], [4], [5], [6], [7], [8], [9], [10]]. The expression of human PDPN (hPDPN) has been reported in several malignant tumors, including oral squamous cell carcinomas [11], esophageal cancers [12], lung cancers [13], malignant mesotheliomas [14,15], osteosarcomas [[16], [17], [18]], chondrosarcomas [17], malignant brain tumors [[19], [20], [21], [22]], and testicular tumors [23]. The expression of hPDPN is associated with malignant progression and cancer metastasis [6,19,24]. Until now, we have developed monoclonal antibodies (mAbs) against human [25], mouse [25], rat [26], rabbit [27], bovine [28], dog [29], and cat [30] PDPNs. Furthermore, an anti-cat PDPN mAb (PMab-52) cross-reacted with tiger PDPN [31]. Although an anti-horse PDPN (horPDPN) mAb, PMab-202 was recently established by immunizing mice with synthetic peptides of horPDPN, it was not useful for immunohistochemical analysis [32]. Sensitive and specific mAbs against horPDPN are necessary to investigate the expression and function of horPDPN. In the present study, we immunized mice with CHO/horPDPN cells and established hybridomas that could produce mAbs against horPDPN. 2.?Materials and methods 2.1. Cell lines CHO-K1 and P3X63Ag8U.1 (P3U1) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The horse kidney cell line, FHK-Tcl3.1, was Rivaroxaban (Xarelto) established at Yamaguchi University [33]. The horPDPN bearing an N-terminal PA16 tag (PA16-horPDPN) was inserted into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) [32]. The PA16 tag comprises 16 amino acids (GLEGGVAMPGAEDDVV) [34]. CHO-K1 cells were transfected with pCAG-Ble/PA16-horPDPN using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA). Stable transfectants were selected by limiting dilution and cultivated in a medium containing 0.5?mg/mL of zeocin (InvivoGen, San Diego, CA, USA). P3U1, CHO-K1, and CHO/horPDPN cells were cultured in Rivaroxaban (Xarelto) Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and FHK-Tcl3.1 was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Inc.) [32]. All media were supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc.), 100 units/mL of penicillin, 100?g/mL of streptomycin, and 25?g/mL of amphotericin B (Nacalai Tesque, Inc.). Cells were grown at 37?C in a humidified environment with an atmosphere of 5% CO2 and 95% ambient air. 2.2. Animals Female BALB/c mice (6 weeks old) 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 the animal experiments. 2.3. Hybridoma production Two BALB/c mice were immunized with CHO/horPDPN cells (1??108), which were intraperitoneally (i.p.) administered together with Imject Alum (Thermo Fisher Scientific Inc.). The procedure included an additional three immunizations followed by a final booster injection administered i.p. 2 days prior to the harvest of spleen cells, making a total of five immunizations. Subsequently, these spleen cells were fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN, USA), and the hybridomas were grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific Inc.). The culture supernatants Rivaroxaban (Xarelto) were screened using flow cytometry. 2.4. Flow cytometry The cells were harvested following brief exposure to 0.25% trypsin/1?mM EDTA (Nacalai Tesque, Inc.). The cells were washed with 0.1% BSA/PBS and treated with primary mAbs for 30?min?in 4?C. Thereafter, the cells had been 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.). Fluorescence data had been gathered using SA3800 Cell Analyzers (Sony Corp., Tokyo, Japan). 2.5. Dedication of binding Rivaroxaban (Xarelto) affinity using movement cytometry FHK-Tcl3 or CHO/horPDPN.1 (2??105?cells) was suspended in 100?L of diluted MULK PMab-219 serially, accompanied by addition of Alexa Fluor 488-conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Rivaroxaban (Xarelto) Inc.). Fluorescence data had been gathered using EC800 Cell Analyzer (Sony Corp.). The dissociation continuous (KD) was acquired by installing the binding isotherms to built-in one-site binding versions in GraphPad PRISM 6 (GraphPad Software program, Inc., La Jolla, CA, USA). 2.6. European blotting Cell lysates (10?g) were boiled in.