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3 protocols using pe cy7 conjugated anti cd45

1

Multicolor Flow Cytometry of Immune Cells

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Single cell suspensions obtained from liver tissues as indicated in 2.4 were stained in FACS buffer with the following anti-mouse antibodies: PE-Cy5 conjugated anti-F4/80 (eBioscience), Alexa Fluor 700 conjugated anti-CD11b (eBioscience), PE-Cy7 conjugated anti-CD45 (eBioscience), FITC conjugated CD4 (BioLegend), BUV395 conjugated CD8a (BD Biosciences), Pacific Blue conjugated CD44 (BioLegend), FITC conjugated F4/80 (BioLegend), FITC conjugated CD3e (BD Biosciences), FITC conjugated Ly-6G (BioLegend), APC-eFluor 780 conjugated CD11c (eBioscience), APC conjugated IFNγ (BioLegend), PerCP-Cy5.5 conjugated Ly-6C (eBioscience) and PE conjugated anti-Ly6G (BioLegend). Acquisition was performed on a LSR Fortessa, LSR Fortessa X-20 or LSRII. Data were analyzed with FlowJo 7.5.5 software (Tree Star).
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2

Mammary Cell Isolation and Characterization

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Mammary cells were obtained as performed in earlier studies [15] (link), [22] (link). In brief, inguinal mammary gland or interscapular BAT samples were dissociated by scissors and then incubated with 5% fetal bovine serum containing collagenase (300 IU/mL, Sigma) and hyaluronidase (100 IU/mL, Sigma) for 60 min at 37°C. Samples were then centrifuged at 500 g for 5 min, and the cell fractions were incubated with 0.25% trypsin-EGTA for 3 min, then resuspended in Dispase (5 mg/mL, Sigma) and DNaseI (50 IU/mL, Takara) for 5 min, and red blood cell lysis (0.64% NH4Cl) for 3 min before filtration through a 40 μm cell mesh. Antibodies were incubated in PBS with 5% FBS for 20 min. The following primary antibodies were used: Percp-cy5.5 conjugated anti-CD24 (eBioscience, Clone M1/69), APC conjugated anti-CD29 (eBioscience, Clone HMB1-1), PE-cy7 conjugated anti-CD31 (eBioscience, Clone 390), PE-cy7 conjugated anti-CD45 (eBioscience, Clone 30-F11). The positive antibody signals were gated based on fluorescence minus one (FMO) control every time. Cell sorting was performed on FACSAria, and the data were read using Flowjo7.6.1 software.
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3

Isolation of Pancreatic Cell Subsets

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Single cell suspensions of 3 week old pancreata were generated with a modified version of a protocol established by Sugiyama and colleagues, 2007 [26] (link). Briefly, pancreatic lymph nodes were removed, and pancreata were minced and sequentially incubated with collagenase D (1 mg/ml in Hanks buffered saline solution, Roche), trypsin (0.05%, Invitrogen), and dispase (2 U/ml, Invitrogen). Dnase1 (100 ug/ml, Sigma) was added during all enzyme incubations. Cells were washed with PBS between the collagenase and trypsin steps, and with FACs buffer (2% FBS, 10 mM EGTA, in PBS) between the trypsin and dispase steps. Suspensions were then filtered through 40 uM mesh, subjected to FC block (1∶200), and incubated with PE-conjugated anti-Cd49f (1∶50, Ebioscience), PE-CY7-conjugated anti-CD45 (1∶500, Ebioscience), and biotin conjugated anti-CD133 antibodies (1∶50, Ebioscience). Streptavidin conjugated to APC was then applied to detect CD133 positive cells (1∶100, Ebisocience). Cells were washed with FACs buffer between block, primary and secondary antibody incubations. DAPI (300 nM) was used as a live cell marker. Cell sorting was performed on a FacsARIAII (Becton Dickinson).
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