Cd24 pe cy7

Mammary glands from 8- to 12-wk-old virgin female mice or mice at lactation day 2 were isolated. The minced tissue was digested in RPMI 1640 with 25 mM HEPES, 5% FBS, 1% PSQ, 300U/mL Collagenase III (Worthington) for 2 hours at 37°C. After lysis of the red blood cells, single cell suspension was obtained by sequential incubation with 0.25% Trypsin-EDTA for 5 min and 0.1 mg/mL DNase I (Sigma) for 5 min at 37°C with gentle pipetting, followed by filtration through 40-μm cell strainers. The antibodies used for flow cytometry were: PE-conjugated CD31, CD45, TER119 (BD PharMingen), CD24-PE/cy7 and CD29-APC (Biolegend). All sorting was performed using FACSAria or FCASJazz (Becton Dickinson). The purity of sorted population was routinely checked and ensured to be >95%. Cells were harvested for quantitative RT-PCR experiments. For insulin treatment experiment, primary mammary epithelial cells were seeded in DMEM (Invitrogen) supplemented with 10% FBS. Adhered cells were treated with 10μg/mL insulin for 24 hours and were harvested for quantitative RT-PCR experiments. Cytosolic triacylglycerol and nonesterified fatty acid in primary mammary epithelial cells were quantified following manufacturer's protocols (Triglycerides kit, Shanghai KEHUA bio-engineering CO., and LabAssay NEFA kit, Wako).

The following antibodies were used for flow cytometric analyses: CD326 (EpCam)
APC-Cy7 (clone G8.8), CD24 PE-Cy7 (clone: M1/69), T1α/podoplanin APC
(clone: 8.1.1.), CD31 Pacific Blue (clone 390), all Biolegend. CD45 V450 (clone
30-F11, BD Biosciences). Multicolor flow cytometry was performed with an LSR
Fortessa® using DIVA software (BD Bioscience). For analytical
measurements
0.5–1 × 10⁶cells were freshly stained with fluorochrome-labeled antibodies for
15 minutes at 4 °C in MACS buffer. The
stained cells were washed and fixed in 4% paraformaldehyde, and resuspended in
MACS buffer.

For cell sorting experiments, 100.10⁶ intestinal epithelial cells were rinsed in FACS buffer (PBS, 3% FCS, 2 mM EDTA), blocked with Human Fc Receptor Binding Inhibitor Antibody (eBioscience) and stained with CD45-BV421 (Bio Legend) and CD24-PeCy7 (Bio Legend) antibodies. Dead cells were excluded with propidium iodure (eBiosciences). During sorting experiments, cells were placed in FACS buffer with RNAse inhibitor (Life Technologies). CD24 marker was used to select EEC, and CD45 was used to get rid of immune cells, especially B cells that also expressed CD24. CD45-negative, CD24-positive EEC were sorted on a jet-in-air flow cytometer (MoFlo Astrios, Summit software, Beckman Coulter). After FACS, an EEC enriched population was obtained containing 1.3.10⁶ cells.
The validation of the EEC sorting was performed in a limited number of independent subjects (n = 7). Gene expression in CD24+ cells was analyzed by RT-qPCR in 3 Ob subjects and protein expression was studied by Western Simple assay on 2 Ob and 2 ObD subjects.

The following antibodies were used in this study:
Fluorescein isothiocyanate (FITC)-conjugated CD31 (BD Biosciences, United States, 553372), CD45 (BD Biosciences, 553080), TER119 (BD Biosciences, 557915); biotinylated CD31 (BD Biosciences, 553371), CD45 (BD Biosciences, 553078), TER119 (BD Biosciences, 553672); CD24-PE/cy7 (Biolegend, United States, 101822), CD29-APC (Biolegend, 102216), Procr-PE (eBioscience, United States, 12-2012-82), PD-L1-PE (eBioscience, 124308), Streptavidin-APC-eFluor 780 (eBioscience, 47-4317-82), and Streptavidin-V450 (eBioscience, 48-4317-82). Antibody incubation was performed on ice for 20–25 min in PBS with 5% fetal bovine serum. All the antibodies were employed at 1:200 dilutions. Before cell sorting and analysis, cells were filtered through 40 μm cell strainers. Single cells gating and sorting were performed using an FACS BD Aria III or Moflo XDP Beckman flow cytometer. All analyses were performed using an LSRFortessa X20. FACS data were analyzed using FlowJo software (Tree Star, United States).

Tumors, inguinal and axillary lymph nodes were harvested 2 weeks after injection. After digestion with DNAse, Collagenase I and Collagenase IV, washing, and counting, 10 million cells per FP were stained with fixable Live/Dead Zombie (BioLegend) and respective antibodies: MHCII-BV421, F4/80-BV510, CD11b-BV605, CD11c-BV650, Ly6C-BV711, CD45R-BV785, CD90.2-BV785, Ly6G-BV785, NK1.1-BV785, CD8alpha-PerCPCy5.5, CD103-APC, CD45-AF700, CD24-PECy7 (Biolegend, eBioscience).
Flow cytometry was performed on a BD LSR Fortessa instrument at the ImmunoX Flow Cytometry CoLab. Analysis of flow cytometry data was done using FlowJo (Treestar) software, and graphs and figures were produced with GraphPad Prism software. All experiments were done in triplicates.