Fix perm reagent

Cells were incubated at 1e6/ml with IgE overnight then washed and plated in new media, as above. 0.66 μl/ml Golgistop (554724, BD Biosciences, San Jose, CA) was added along with AF594-CD107a antibody, clone 1D4B (121624, RRID:AB_2616690, Biolegend, San Diego, CA), then cells were stimulated with 100 ng/ml DNP-BSA and/or 50 ng/ml SCF (250-03, Peprotech) for 4 h at 37 C. Cells were then collected, washed, and fixed/permeabilized with Fix/Perm reagent (554714, BD Biosciences). Cells were then stained with BV421-CD117 (clone 2B8, 105827, RRID:AB_10898120) and PE-FceR1a (clone MAR-1, 134307, RRID:AB_1626104) (Biolegend) in perm/wash buffer for 20 min at 4 C, washed, and analyzed by flow cytometry for CD117/FceR1a^+/+ CD107^+/− expression.

Lymphocytes were isolated by passage through a 100 μm cell strainer (BD Biosciences, San Jose, CA). Single cell suspensions were subjected to red blood cell lysis by resuspension in ACK buffer. Following blocking of cell surface Fc receptors with anti-CD16/32, lymphocytes were surface stained using anti -CD4, -B220, -PD-1, -CXCR5. Cell viability was determined using Live/dead violet. Stromal cells were identified by staining with anti -CD45-PE, -CD31-PE-Cy7 in stain buffer (20 min, 4°C, Table 2). After extensive washing to remove unbound antibodies, cells were subjected to fixation and permeabilization using Fix/Perm reagent according to manufacturer’s instructions (BD Biosciences). Intracellular staining was performed using rabbit anti-β2AR (Abcam) [33 (link)–35 (link)] followed by donkey anti-rabbit BV421 (Biolegend, San Diego, CA) and goat anti-CXCL13 (RnD Systems, Minneapolis, MN) followed by donkey anti-goat Alex Fluor 647 (Invitrogen). After extensive washing to remove unbound antibodies, flow cytometry was performed on a LSRII running DIVA 6.0 (BD Biosciences). Data was analyzed using FlowJo v10.0 (Treestar, Ashland, Oregon).

Flow cytometric analysis was performed as previously described [19 (link)]. Briefly, harvested BMDM were incubated in 1 μg/mL of anti-mouse Fc receptor antibody in 100 mL PBS containing 0.5% BSA plus 0.02% NaN₃ (FACS buffer) for 15 min on ice. Subsequently, single-cell suspensions were stained for 15 min at 4°C with blue-fluorescent reactive dye, L23105 (Life Technologies) to discriminate dead cells. After washing, 1-3 × 10⁶ cells were surface-stained in FACS buffer for 15 min at 4°C with antibodies recognizing CD11b (Alexa Fluor 700, BioLegend), F4/80 (Brilliant Violet 785, BioLegend), CD86 (Brilliant Violet 421, BioLegend), PD-L1 (PE-Cy7, BioLegend), and PD-L2 (PE, BioLegend). Surface-stained cells were washed three times with FACS buffer and treated with Fix/Perm reagent according to the protocol of the cytofix/cytoperm kit (BD Biosciences, San Jose, CA, USA). The cells were intracellularly stained in FACS buffer containing anti-Nos2 (PE, eBiosciences) and anti-h/m arginase 1 (APC, R&D systems) for 30 min at 4°C and further collected on an LSR II cytofluorometer (BD, Franklin Lakes, NJ). Stained cells were gated according to size (SSC-A) and forward scatter (FSC-A) to eliminate debris. Doublets were excluded from the analysis by using forward scatter height (FSC-H) and FSC-A. Data analysis was performed using FlowJo Software (FlowJo, LLC).