Anti 7 aad

Cell staining was performed in the presence of purified rat anti-mouse CD16/32 antibody (BD). Antibodies used for staining were as follows; anti-Ly6G-PE (1A8, BD), anti-CD11b-APC (M1/70, eBioscience), anti-Ly6G-APC-Cy7 (1A8, BD), anti-Ly6C-APC-Cy7 (AL-21, BD), mouse biotin-conjugated anti-CD45 (30-F11, eBioscience), anti-streptavidin-V500 (BD), and anti-7-AAD (BioLegend).
For intracellular staining for phosphorylated p38 MAPK, 1 × 10⁶/ml of bone marrow-derived neutrophils were stimulated with 1 μg/ml of R848 (Enzo Life Sciences) for 30 min in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum (FBS), 100 μg/ml of L-glutamine (Sigma-Aldrich), 100 U/ml of penicillin (Sigma-Aldrich), 100 μg/ml of streptomycin (Sigma-Aldrich), and 50 μM 2-mercaptoethanol (Sigma-Aldrich). Stimulated neutrophils were stained with anti-CD11b-APC (M1/70, eBioscience) and anti-Ly6G-APC-Cy7 (1A8, BD) antibodies in the presence of purified rat anti-mouse CD16/32 antibody (BD), fixed with Lyse/Fix Buffer (BD), and permeabilized by BD Phosflow Perm Buffer II (BD). Then, neutrophils were intracellularly stained with Alexa Fluor 488 Mouse IgG1κ isotype control (BD) or Alexa Fluor 488 mouse anti-p38 MAPK (pT180/pY182) (BD).
Flow cytometric analysis was performed using MoFlo XDP and data were analyzed using FlowJo Software (Tree star).

Human islet-depleted cell fractions were obtained from organ donors deceased due to acute traumatic or anoxic death by Prodo Laboratories, Inc, and were shipped overnight to our laboratory (n = 21, see donor information in Supplementary Data 1). The flow sorting procedures were performed as previously described with some modifications^{14 (link)}. Briefly, exocrine tissue cells were incubated with FITC-conjugated UEA-1 (0.25 μg/ml, Vector Laboratories, Newark, CA, FL-1061-5) for 10 min. at 4 °C and washed with PBS. After washing, the cells were digested with TrypLE^TM Express (Life Technologies, Grand Island, NY, 12605-028) for 5−8 min at 37 °C. Cells were collected by centrifugation and washed with FACS buffer (10 mM EGTA, 2% FBS in PBS). After washing, cells were stained with Pacific blue-conjugated anti-CLA (BioLegend, San Diego, CA, 321308) and anti-7AAD (BioLegend, San Diego, CA, 420404) for 15 min at 4 °C. Cell pellets were collected by centrifugation and washed with PBS after staining. The cells were sorted using a FACSAria^TM II (BD Biosciences, San Diego, CA) and collected in 100% FBS. After sorting, cells were washed with serum-free Advanced DMEM/F-12 media (Life Technologies, Grand Island, NY, 12634-010).