Armenian hamster igg isotype control apc

Cells were counted and Fc receptors were blocked using anti‐CD16/CD32 (clone 2.4G2) (catalog no. 553141) from Becton‐Dickinson (Franklin Lakes, NJ, USA) to prevent non‐specific binding. Cells for immunofluorescence staining were labelled with monoclonal antibodies: anti‐CD3e FITC (clone 145‐2C11) (catalog no. 11‐0031‐86) or Armenian hamster IgG isotype control FITC (clone eBio299Arm) (catalog no. 11‐4888‐81), anti‐CD11c APC (clone N418) (catalog no. 17‐0114‐82) or Armenian hamster IgG isotype control APC (clone eBio299Arm) (catalog no. 17‐4888‐82), and anti‐B220 PE (clone HIS24) (catalog no. 12‐0460‐82) or mouse IgG2b kappa isotype control PE (clone eBMG2b) (catalog no. 12‐4732‐82) from eBioscience (Carlsbad, CA, USA) and anti‐CD4 PerCP (clone RM4‐5) (catalog no. 553052) and rat IgG2a, κ isotype control PerCP (clone R35‐95) (catalog no. 553933) from BD Pharmingen (San Diego, CA, USA) at 4 °C for 30 min. Excessive antibodies were washed off and labeled cells were analyzed using a flow cytometer (BD ACCURI C6). Data were analyzed usinf flowjo (Tree Star Inc., Ashland, OR, USA) and prism (GraphPad Software Inc., San Diego, CA, USA).

For the analysis of MRGPRX2 expression, LAD2 cell suspensions were seeded at a density of 2 × 10⁵ cells/mL, and incubated with 5 µM of SP for 1 to 60 min at 37°C and 5% CO₂. Cells were washed twice with 0.1% BSA-PBS buffer at 200 × g for 5 min, resuspended in 0.1% BSA-PBS buffer, and incubated for 1 h in the dark at 4°C with anti-human MrgprX2-PE (Biolegend, San Diego, CA, USA); or anti-human FcϵRIα-APC (eBioscience, Invitrogen Carlsbad, CA, USA); washed twice with 0.1% BSA-PBS buffer at 200 × g for 5 min, and fixed with 5% formalin neutral buffered solution (Sigma-Aldrich, Oakville, ON, CAN), for 15 min at RT followed by the addition of 3% BSA-PBS buffer, mixed and centrifuged at 200 × g for 10 min at 4°C, cells were then resuspended in 0.1% BSA-PBS buffer, and analyzed on a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA). Mouse IgG2b, k isotype control-PE, and Armenian hamster IgG isotype control-APC (eBioscience, Invitrogen Carlsbad, CA, USA) were included as negative controls. Expression of MRGPRX2, and FcεRIα were analyzed using FlowJo v.10.8 software (BD Life Sciences, Ashland, OR, USA) and compared to control values. Results are reported as histograms and mean fluorescent intensity (MFI ± SEM).