Biotinylated anti fcεri clone mar 1

BMMCs expressing Siglec-6 were plated in 96-well round bottom tissue culture plates at 5 × 10⁴ per well and centrifuged for 2 min at 400 g prior to resuspending with biotinylated anti-FcεRI (clone MAR-1, Biolegend) at 250 ng mL⁻¹ plus biotinylated isotype-control mouse antibody (MOPC21, mouse IgG1, Allakos) or biotinylated Siglec-6 mAb (AK04 mouse IgG1, Allakos) at 5 μg mL⁻¹ at 4 °C for 2 min. Cells were washed in PBS and then incubated in PBS with 10 μg mL⁻¹ neutravidin (Thermo) for 2 min. After an additional PBS wash, cells were resuspended in 200 μl 37 °C complete medium and incubated for 20 min at 37 °C for flow analysis or for 1 or 6 h for analysis of histamine or cytokine levels in the supernatant. For flow cytometry, cells were resuspended in 100 μl cold FACS buffer (PBS/1%BSA) containing 100 ng anti-CD63-PE/Cy7 antibody (clone NVG-2, Biolegend), 3 μl 7-AAD (Becton Dickinson) as viability marker and 0.2 μl mouse Fc block (BD). The percent of CD63 expressing cells was determined by flow cytometry on a Novocyte Quanteon (Agilent).

S8-BMMC were plated in 96-well round bottom tissue culture plates at 5x10⁴ per well and centrifuged for 2 min at 400g prior to resuspending with biotinylated anti-FcεRI (clone MAR-1, Biolegend) at 250ng/mL plus biotinylated isotype-control mouse antibody (MOPC21, mouse IgG1, Allakos) or biotinylated Siglec-8 mAb (2E2, mouse IgG1, Allakos) at 5μg/mL at 4°C for 2 min. Cells were washed in PBS and then incubated in PBS with 10μg/mL neutravidin (Thermo) for 2 min. After an additional PBS wash, cells were resuspended in 200μl 37°C complete medium and incubated for 20 min at 37°C for flow analysis or for 1 or 6 hours for analysis of histamine or cytokine levels in the supernatant. For flow cytometry, cells were resuspended in 100μl cold FACS buffer (PBS/1%BSA) containing 100ng anti-CD63-PE/Cy7 antibody (clone NVG-2, Biolegend), 100ng anti-CD107a-PE (clone 1D4B, Biolegend), 3μl 7-AAD (Becton Dickinson) as viability marker and 0.2μl mouse Fc block (BD). The percent of CD63 and CD107a expressing cells was determined by flow cytometry on a Novocyte Quanteon (Agilent). For cytokine quantification, 25μl of supernatant was analyzed using Meso Scale Discovery’s U-plex kit (customized for the quantification of the indicated cytokines). Histamine levels were determined using an enzyme immunoassay kit (Beckman Coulter).