Ma900 flow cytometer

Yeast surface display was performed as previously described (90 (link)). Briefly, the human IL-2 sequence (GenBank: AAB46883.1) was modified using site saturation mutagenesis, error-prone PCR, or synthesis of sequences with directed substitutions. Amplicons were cotransformed with a linearized expression vector into EBY100 yeast and cultured as previously described (91 (link)). Yeasts expressing the IL-2 variants were assessed in 2 distinct ways. To measure expression levels (a surrogate for aggregation and stability), yeast were stained with fluorescent antibodies against the c-Myc epitope tag present at the C-terminus of the IL-2 protein. Additionally, yeasts were incubated with a recombinant CD122/CD132 heterodimer, and binding was detected with goat anti–human Fc conjugated with a fluorophore. Yeasts expressing high levels of IL-2 and/or reduced binding to CD122/CD132 were sorted using a Sony MA900 flow cytometer. The IL2 gene sequences within the enriched libraries were PCR amplified and sequenced with an Illumina MiSeq 2 × 75 PE (Genewiz).

Pancreatic tissue isolation was performed as previously^{36 (link)} with minor modifications. Briefly, pancreas was minced into small pieces, and transferred to 3 mL 0.5 mg/mL Collagenase P (Roche, 11213873001) dissolved in HBSS together with 5 mM glucose. Then the digestion medium was put into the water bath at 37 °C 5 min for acclimatization and gently shaking another 5 min for digestion. To stop the digestion, 10 mL DMEM containing 1% FBS was added to the medium. Tissues were pipetted up and down for further dissociation and filtered by the 70-μm filter. Then cells were centrifuged for 5 min at 1200 rpm and incubated with 1 mL red blood cell lysis buffer (eBioscience, 00-4333-57) at room temperature for 5 min. 10 mL cold PBS containing 1% FBS was added to stop digestion, followed by centrifugation. 1% Fc in PBS was added for 5 min to block, then the isolated cells were stained with fluorochrome-conjugated antibodies including CD45-APC (eBioscience, 17-0451-82, 1:400), CD31-APC (eBioscience, 17-0311-82, 1:40), CD140a-APC (eBioscience, 17-1401-81, 1:100), CD326-APC (eBioscience, 17-5791-82, 1:200) for 30 min at 4 °C. After washing with cold PBS, cells were resuspended in PBS containing DAPI (1:1000). Then flow cytometry experiments were performed using the Beckman Cytoflex LX or sorted using the Sony MA900 flow cytometer.

Islets were dispersed and then stained with fluorescence-tagged antibodies to detect GFP + cells or CD11c + F4/80+ macrophages. These cells were analyzed or sorted out by MA900 flow cytometer (SONY). All antibodies were used with 1:200 dilution. Data were analyzed using Flowjo software.

The liver and heart were perfused to get single cells which were then stained with fluorescence-tagged antibodies. KCs were CD45⁺Clec4f⁺F4/80⁺ cells, and cardiac macrophages were CD45⁺CD11b⁺F4/80⁺ cells. These cells were analyzed by an MA900 flow cytometer (SONY). Data were analyzed using Flowjo software. Clec4f (Cat. No. 156804), F4/80 (Cat. No. 123114), CD11b (Cat. No. 101235), and CD45 (Cat. No. 103116) antibodies were received from Biolegend, and Vsig4 antibody (Cat. No. 17-5752-82) was from ThermoFisher Scientific. The concentration of antibodies (0.25µg per 10⁶ cells) for sample staining were used.