P nitrophenyl n acetyl β d glucosamine

hMCs were stimulated with L. monocytogenes (100 µL, 5 × 10⁵ cells/mL) for 2 hours. After incubation, supernatants were harvested and cell pellets were lysed in 1% Triton X‐100. β‐Hexosaminidase activity was measured in supernatants as well as in cell pellets by adding the substrate p‐nitrophenyl N‐acetyl‐β‐d‐glucosamine at 1 mmol/L (Sigma‐Aldrich) in 0.05 mol/L citrate buffer (pH 4.5) for 2 hours at 37°C in a 5% CO₂ atmosphere.⁵⁶ The reaction was stopped by using 0.05 mol/L sodium carbonate buffer (pH 10). OD was measured at 405 nm. Degranulation was assessed as the percentage release of total β‐hexosaminidase.

All cell culture and Lipofectamine 2000 transfection reagents were obtained from Invitrogen (Gaithersburg, MD, USA). Amaxa Nucleofector kit (Kit V) was purchased from Lonza (Gaithersburg, MD, USA). Phycoerythrin (PE)-conjugated (Cat.#359004) and purified unconjugated anti-MRGPRX2 antibodies (Cat.#359002) were from BioLegend (San Diego, CA, USA). Donkey anti-mouse Alexa Fluor 488 (Cat.#A21202) and 647 (Cat.#A31571) conjugated IgG secondary antibodies were from Invitrogen (Gaithersburg, MD, USA). p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) was from Sigma-Aldrich (St. Louis, MO, USA). Fura-2 acetoxymethyl ester was from Abcam (Cambridge, MA, USA). SP was purchased from AnaSpec (Fremont, CA, USA). Pertussis toxin (PTx) was from List Biological Laboratories (Campbell, CA, USA). Plasmid encoding hemagglutinin (HA)-tagged human MRGPRX2 in pReceiver-MO6 vector was obtained from GeneCopoeia (Rockville, MD, USA). MRGPRX2 Y279A mutant in HA-tagged plasmid was reported previously [28 (link)]. MRGPRX2-Tango plasmid (Addgene no. 66440) was a gift from Dr. Bryan Roth. MRGPRX2 Y279A mutant in Tango plasmid was generated by Penn Genomics Analysis Core (Philadelphia, PA, USA).