Cd16 cd32 fc block

Prior to antibody staining, samples were blocked with Fc-block CD16/CD32 (Biolegend, 101320, 1:50) in FACS buffer for 10 min on ice to block nonspecific binding. For cell surface staining, cells were resuspended in the appropriate antibody cocktail and incubated for 30 min on ice protected from light. Samples were centrifuged and washed with a FACS buffer. For intracellular staining, cells were then collected and centrifuged for 5 min 350 g. Cells were fixed, permeabilized and stained for transcription factors using the True-Nuclear Transcription Factor Buffer Set (Biolegend, Cat# 424401) according to the manufacturer’s instructions. Viability was assessed by staining with DAPI. Information and dilution for antibodies used for flow cytometry are listed in Supplementary Table 1. For fibroblast sorting cells, PDGFRα⁺ GFP^- tdTomato⁺ were selected and sorted directly into a lysis solution from Arcturus PicoPure RNA Isolation Kit (Thermofisher). For sample validation, cells were plated in poly-L-lysine treated (Sigma) glass coverslips in 12-well plates and cultured for 24 h prior to IF. Immune cell profiling by flow cytometry was carried out by analysing 50.000 live singlets in each sample. All fluorescent data were analysed using BD FACSDiva Software (BD Bioscience).

For conventional fluorescence microscopy analysis, BMDCs and B‐cells were stained with saturating amounts of fluorophore‐conjugated scF_V (e.g., 10 μg ml⁻¹ 14.4.4 scF_V‐AF647) or mAbs (14.4.4 mAb‐AF647, 11–5.2 mAb‐AF647, and IgG (H + L)‐AF647) for 30 min on ice, and washed twice with 10 ml 1× HBSS (Gibco) supplemented with 1% FBS (Biowest), 2 mM CaCl₂, and 2 mM MgCl₂ (Merck) prior to seeding the cells onto ICAM‐1‐coated glass slides. Cells were treated with the Fc block CD16/CD32 (BioLegend) at 20 μg ml⁻¹ for 15 min prior to the addition of mAbs. For experiments involving chemical fixation, cell samples were treated with 4% PFA (Thermo Fisher Scientific) in 1× PBS for 15 min at room temperature and subsequently washed in 1× HBSS (Gibco) supplemented with 1% FBS (Biowest), 2 mM CaCl₂ and 2 mM MgCl₂ (Merck) to stop the fixation reaction.
Mid‐sections of BMDCs or B‐cells were recorded as z‐stacks (50 images with 0.3 μm spacing, total depth of z‐stack: 15 μm) and deconvolved with the ImageJ plugin iterative deconvolve 3D, which is based on the DAMAS (Deconvolution Approach for the Mapping of Acoustic Sources) algorithm (Dougherty, 2005 ). The point spread function in x, y, and z used to deconvolve the images was measured with single fluorescent beads (TetraSpeck Fluorescent Microspheres).