Prior to FACS staining, tumor spheroids were dissociated into single cells by trypsination for 10 min at 37 °C, followed by vortexing for 10 s. To analyze cell cycle distribution, tumor single cells were fixed in 70 % ice-cold ethanol, washed with PBS + 2 % FCS, and treated with 100 μL RNAse (1 mg/mL) at 37 °C for 20 min. Cells were stained with 10 μL propidium iodide (1 mg/mL) for 30 min. FACS analysis was performed on a FACS Canto II instrument using Diva Software (BD Biosciences). Proliferation was analyzed by FACS after staining tumor single cells with anti-Ki67 PEVio770, anti-CD45 FITC, and anti-EpCAM PE antibodies (all Miltenyi Biotech).
Anti cd45 fitc
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD45-FITC is a fluorescently-labeled antibody that binds to the CD45 antigen expressed on the surface of various hematopoietic cells, including leukocytes. This product can be used for the identification and enumeration of CD45-positive cells in flow cytometric analyses.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd34 pe, by Miltenyi Biotec (3 mentions)
Anti sca1 apc, by Miltenyi Biotec (2 mentions)
Accutase, by Innovative Cell Technologies (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti epcam pe, by Miltenyi Biotec (1 mentions)
Facscanto 2, by BD (1 mentions)
Diva software, by BD (1 mentions)
Anti fitc magnetic microbeads, by Miltenyi Biotec (1 mentions)
Human tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Trustain fcx, by BioLegend (1 mentions)
Propidium iodide, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Anti cd8 v500, by BD (1 mentions)
Anti cd3 alexa 700, by BD (1 mentions)
Anti cd14 percp, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Anti cd4 pacific blue, by BD (1 mentions)
Trypsin, by Cambrex (1 mentions)
Facsaria 2, by BD (1 mentions)
14 protocols using anti cd45 fitc
1
Cell Cycle Analysis of Tumor Spheroids
2
Sorting and Isolating ASCs and FAP from SVF
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ASC sorting: ScAT- or PGAT- derived SVF were depleted in CD45+ and CD31+ cells using anti-CD45-FITC (Miltenyi, 130-116-535) and anti-CD31-FITC antibodies (Miltenyi, 130-123-675) followed by anti-FITC magnetic microbeads (Miltenyi, 130-048-701) using an autoMACS® Pro Separator (MACS Cell Separation, Miltenyi Biotec SAS) according to the manufacturer’s instructions. CD45−/CD31− cells were then positively sorted for Sca-1 with anti-Sca-1 magnetic microbeads (Miltenyi, 130-106-641). Isolated ASCs were used for RNAseq experiments, for muscle injection in platelet-depleted or lipectomied animals (see methods), or for in vitro platelet/ASCs interaction challenges (see methods). FAP sorting: muscle-derived SVF were treated as above and used for RNAseq experiments, affymetrix analysis (ctrl or injured), and DNA extraction.
3
Single-cell Immune and Non-immune Cell Isolation
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Tissue samples were enzymatically dissociated using a human Tumor Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations, and red blood cells were lysed using 1X RBC solution (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) for 5 min at room temperature before quenching with cold phosphate-buffered saline. Subsequently, single cell suspensions were Fc-blocked using TruStain FcX (1:125 dilution; BioLegend, San Diego, CA, USA) for 5 min on ice. Surface CD45 was stained with anti-CD45-FITC (1:50; #REA747, Miltenyi Biotech) for 20 min at 4 °C. Washed cells were stained with propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, cat: 556463) and with DRAQ5 (#62251, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:5000 in cold 1% bovine serum albumin in phosphate-buffered saline and stained for 10 min at room temperature. Single cells were sorted for live, nucleated CD45+ and CD45− cells to enrich for immune and non-immune cells.
4
Identification of Infiltrating Macrophages and Microglia
5
Leukocyte BST2 Expression Analysis
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Surface BST2 expression was detected on different subsets of leukocytes, defined by anti-CD3-Alexa700, anti-CD4-Pacific Blue, anti-CD8-V500, anti-CD14-PerCP (all from BD Biosciences, Heidelberg, Germany) and anti-CD45-FITC (Miltenyi, Bergisch-Gladbach, Germany), by using an anti-BST2-APC conjugated antibody (RS38E, Biolegend). Labeled cells were fixed with 3 % formalin and analyzed on a BD LSRII flow cytometer (BD Biosciences, Heidelberg, Germany). The data files were evaluated using FlowJo Version 8.7 (Tree Star, Ashland, USA). Median fluorescence intensity (MFI) for granulocytes, monocytes and lymphocytes were determined.
6
Flow Cytometry Analysis of Adipose-Derived Cells
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Cells (from lipoma and normal adipose tissue) were detached using trypsin-EDTA (200 mg/L EDTA, 500 mg/L trypsin; Cambrex, Milan, Italy). Cells were incubated with primary antibody for 30 min at 4°C, washed twice in PBS and incubated with a secondary antibody. Alternatively, cells were incubated directly with fluorescent-conjugated antibodies for 30 min at 4°C, washed and resuspended in 0.6 ml of PBS. Samples were analysed at day 0 (day of surgery) and day 30 by flow cytometry using a FACS Aria II (Becton&Dickinson, Mountain View, CA). The antibodies used in this study were: anti-CD34 PE and anti-CD45 FITC (all purchased from Miltenyi-Biotech, Calderara di Reno, Bologna, Italy). CD34+ and CD34- cells were sorted for experiments. The purity of sorted population was routinely 90%.
7
Fibroblast-Macrophage Co-Culture Proliferation
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Mammary fibroblasts and macrophages were co-cultured in 96-well plates at a range of concentrations (0,1 × 103, 1 × 104 and 3 × 104), and an EdU incorporation assay was performed on day 7. EdU (10 mM) was added to the cells for 2 h, after which the cells were harvested, stained with the live/dead exclusion marker Ghost-Dye-Violet450 (TONBO, Cat.13-0863), and with anti-CD45-FITC (Miltenyi Biotec, Cat.130-110-658). EdU incorporation was detected using the Click-iT Plus EdU Flow Cytometry Assay Kit according to the manufacturer’s instructions (ThermoFisher, Cat. C10634). Samples were then acquired using a CytoFlex-S (Beckman Colter), macrophages were gated based on positive staining for CD45, and fibroblasts were called based on negative staining for this marker. Analysis was performed with FlowJo 10.7.1.
8
Isolation and Characterization of Tumor-Associated Fibroblasts
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4T1-GFP cell line was orthotopically injected into the mammary fat pad of 8 week old BALB/c female mice (1 × 105 cells in 50 µl PBS). After 4 weeks, the mice were sacrificed and the tumors were harvested and dissociated in gentle MACS dissociator with enzymatic solution containing 3 mg/ml collagenase (Sigma Aldrich, 11088793001) and 0.1 mg/ml Dnase in RPMI 1640 using the standard program for solid tumors. To receive single cell suspension, the digested cell suspension was filtered through 70 µ strainer with ice-cold PBS. The cells were pelleted and lysed in red blood cell lysis buffer and depleted of CD45+ and EpCAM+ cells as described above. Cells were stained for anti-CD45 FITC (Miltenyi 130-110-658), anti-CD31 FITC (Miltenyi 130-123-675), and anti-EPCAM FITC (Miltenyi 130-117-752), anti-PDPN APC (Biolegend 127410) and anti-Ly6C Pacific Blue (Biolegend 128014). See Supplementary Table 3 for antibodies information. Dead cells were excluded using PI Staining (Sigma Aldrich, P4170). CAF were gated based on PI-, CD45-, CD31-, EpCAM-, PDPN+. For sorting the CAF subpopulation, we used LY6C+ as a marker for iCAF and LY6C- as a marker for myCAF. The cells were sorted using a FACSAria Fusion (BD Biosciences) into FACS tubes containing 1 ml complete DMEM media. The maximum tumor volume of 1000 (mm)3 was not reached in any experiment.
9
Isolation of Bone Marrow Stromal Cells
10
Cell Sorting and RNA Preservation
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