Extended Data Fig. 4: Gal-3 upregulation in islet and peritoneal macrophages following apoptotic β-cell uptake ex vivo.

a. Isolation of islet macrophages using CD11c⁺ magnetic beads (MACS) (non-autoimmune C57BL/6 J mice). Flow cytometric analysis on dispersed islet cells before enrichment (left), and MACS-enriched Live, CD45⁺ F4/80⁺ CD11c⁺ macrophages (right). b. Schematic of the assay when primary islet macrophages co-cultured with live or apoptotic β-cells (UV-irradiated Min6 cells). c. Flow cytometric evaluation of e-Mac frequencies in the co-culture experiment (b). Quantification is shown on the right. n = 4(UT), n = 2(Live), n = 8(Apo) biological replicates over N = 4 independent experiments for “UT” and “Apo” and N = 2 for “Live”. Two-tailed unpaired t-test with Welch’s correction. d. Flow cytometric analysis showing peritoneal macrophages. Peritoneal lavage cells were collected from C57BL/6 mice, plated for 2 h, washed to remove unbound cells, and analysed by flow cytometry 24 h after the beginning of the incubation. e. Flow cytometric analysis showing engulfment of apoptotic β-cells by peritoneal macrophages. Apoptotic β-cells (Min6 cell line) were labelled with pHrodo red dye and co-cultured with pMacs for 2 h; unbound apoptotic cells were then washed away, and incubation continued. The macrophage-to-β-cell ratio was 1:3. Flow cytometry was done 16 h after beginning of the incubation. f. Flow cytometric analysis of peritoneal macrophages after co-culture with live or apoptotic β-cells (Min6). Two negative controls were used: cytochalasin D (CytoD, 1 µM) – inhibitor of cytoskeletal reorganization that prevents corpse uptake; and annexin V (20 µg/ml) that binds phosphatidylserine (PtdSer) and masks it from scavenger receptors on the phagocytic cells. g. Quantification of (f). n = 6 (No β-cell), n = 6 (Live β-cell), n = 6 (Apo β-cell, alone), n = 3 (Apo β-cell, +CytoD), n = 3 (Apo β-cell, +Ann V) biological replicates; N = 2. Statistics - two-tailed unpaired t-test. Data are representative of N = 3 independent experiments (a, b). The diagrams in a and b were created in BioRender. Ravichandran, K. (2025) https://BioRender.com/oc6ssn7; Ravichandran, K. (2025) https://BioRender.com/laier1m.

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