Extended Data Fig. 2: Spermine is enriched in mast cell extracellular granules (MCEGs).
From: Mast cell extracellular granules are bioactive condensates assembled by heparin and polyamine
a, Fluorescence images of BMMCs expressing mCherry-Tryptase (red) or mCherry-TNFα (magenta) loaded with 20 μM BODIPY-spermine (green) for 1 h. Right panels show fluorescence intensity profiles along the yellow dashed line. Scale bar, 5 μm. b, Flow cytometry analysis of spermine (BODIPY-spermine) and heparin (AF647-WGA) in MCEGs from BODIPY-spermine-preloaded mCherry-tryptase or mCherry-TNFα expressing BMMCs. Histograms show proportions of heparin-only, spermine-only, double positive (Hep+ & Spm+), or negative (Hep- & Spm-) MCEGs within total (left) or mCherry-mediator-retained (right) MCEG population. N = 3. c, MCEGs stability in heparin- and spermine-free buffers. MCEGs released from BODIPY-spermine-loaded BMMCs were incubated in tyrode’s buffer at 37oC. Flow cytometry measured MCEG counts and mean fluorescent intensity (MFI) of heparin (labeled with sRd101-avidin) and BODIPY-spermine over time, normalized to Day 0 (set as 100%). N = 3. d, FRAP of reconstituted TNFα condensates. Right panel shows fluorescence recovery of Cy3-TNFα (1.25 μM) in reconstituted condensates (50 μM heparin, 5 mM spermine). N = 23 individual condensates. Scale bar, 2 μm. e. FRAP of MCEGs containing BODIPY-spermine (green), sRd101-avidin/heparin (yellow), or mCherry-TNFα (magenta). MCEGs were separated by low-speed centrifugation and photobleached in IgE/Ag stimulation supernatant. Right panel shows fluorescence recovery. N = 28 (sRd101-avidin/heparin), N = 39 (spermine), or N = 14 (TNFα). Scale bar, 5 μm. Data are presented as Mean ± SD. One representative experiment from 3 independent experiments was shown.
