Ic003r

RASF were primed with TNF (10 ng/mL) (PeproTech, Hamburg, Germany) or left untreated for 72 h in RPMI medium with 2% FCS. Then, cells were analyzed for surface and intracellular expression of cannabinoid receptors. The following antibodies were used: CB₁ (FAB3834R, 0.2 mg/mL, 1:10, R&D Systems/Biotechne, Wiesbaden, Germany), CB₂ (FAB36551G, 0.2 mg/mL, 1:40, R&D Systems/Biotechne), Isotype MsIgG2a-Alexa 488 (IC003G, 5 µL/test, R&D Systems/Biotechne), and Isotype MsIgG2a-Alexa 647 (IC003R, 5 µL/test, R&D Systems/Biotechne5 µL/test); RASF were detached from culture dishes with citrate buffer (135 mM KCl, 15 mM Na₃C₆H₅O₇) and centrifuged at 300× g. Cells were resuspended in PBS with 10% FCS and incubated with antibodies for 30 min in the dark at room temperature. For intracellular staining, the inside stain kit was used (#130-090-477, Miltenyi biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.

Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (Clone # 535919) (R&D systems, FAB9332R), AF-488 mouse anti-human ACE2 (Clone # 171607) (R&D systems, FAB9333G) (0.4 µg/10⁶ cells), AF-647 isotype control mouse IgG2b (Clone # 20102) (R&D systems, IC003R) or AF-488 isotype control mouse IgG2bAF488 (Clone # 20102) (R&D systems, IC003G) for 45 min on ice, followed by washing twice with 2% EV-free FBS/PBS. Finally, the cells were diluted in 2% EV-free FBS/PBS and analyzed on a BD-LSR II flow cytometer (BD Biosciences). Data were analyzed by BD FACSDiva softwares v8.0.2 or v8.0.3 or Flow Jo v10.6.2.