Anti mouse cd16 32 mab

In addition to the analysis of ETAR and ETBR by real-time PCR (Supplementary Fig. 2), the expression of ETAR by MDA-MB-231 and 4T1 cells was confirmed at the protein level by flow cytometry using a modification of a previously published protocol^{83 (link)}. Briefly, 3 × 10⁵ cells suspended in staining buffer (PBS/1% FCS/ 0.1% NaN3) were pre-incubated with either Human Trustain Fcx or anti-mouse CD16/32 mAb (both from Biolegend, San Diego, CA, USA) to block FcγIII/II receptor sites on MDA-MB-231 or 4T1 cells, respectively. The cells were then stained with Zombie Aqua Viability dye (Biolegend). After washing, the cells were stained with ETAR-specific rabbit polyclonal antibody (Cat# MBS9203839; MyBiosource, San Diego, CA) for 30 min. After 3 rounds of washing in staining buffer, cells were incubated with FITC-conjugated goat anti-rabbit IgG secondary antibody (cat# Ab97050; Abcam, Cambridge, UK). Data were collected on 10,000 cells per sample using FACS Celesta (BD) and viable cells were analyzed using FlowJo software (BD Bioscience).

After the mice were sacrificed, the lungs were removed, washed with PBS, cut into small pieces using a scalpel, transferred into GentleMACS C-tubes (Miltenyi Biotec, Germany), and processed using the lung dissociation kit and a GentleMACS dissociator (Miltenyi), according to the manufacturer’s instructions. Homogenized lungs were passed through a 70-mm nylon mesh to obtain a single-cell suspension. The resultant cells were counted using an automated cell counter (EVE, NanoEnTek, Seoul, South Korea) and processed for flow cytometry, as detailed previously^{94 (link),95 (link)}. Briefly, cells were pre-incubated with anti-mouse CD16/32 mAb (Biolegend) to block FcγIII/II receptor sites and then stained with Zombie Aqua Viability dye (Biolegend). After washing, the cells were stained with a mixture of fluorochrome-conjugated antibodies using the following panel of mAbs: CD45-Alexa Fluor 700, CD11b-Alexa Fluor 488, Ly6G-BV605, Ly6C-APC-Cy7, F4/80-PE, and MHC II-BV785 (all from Biolegend). Data were collected on 30,000 cells per sample using FACS Celesta (BD) and analyzed using BD FACS Diva software (BD Bioscience).