Cell staining for flow cytometry (FACS) analysis and sorting was performed at 4°C, in the dark, for 20–30 min, with the exception of anti-CCR7 antibody (BioLegend) staining that required incubation at 37°C for a minimum of 30 min. To exclude dead cells, either Hoechst 33258 (Sigma-Aldrich) or viability dye eFluor 506 (eBioscience) was used. FACS analysis of TECs and DCs was performed using BD LSR II and BD FACSymphony A5 cytometers, respectively. A BD FACSAria IIu sorter was used for cell sorting. BD FACSDiva software and FlowJo V10 software (Treestar) were used for FACS data analysis. For the purpose of tSNE analysis, the same amount of CD11c+ TdTOM+ cells from each model was concatenated by using the FlowJo concatenate function. The final tSNE was calculated by FlowJo opt–SNE plugin. Total TEC counts in Figure 1e were obtained by analyzing the whole fraction of CD45– MACS-enriched cells. Calculation of the ratios in Figures 1g and 2g is described in the ‘Results’ section and figure legends. The entire list of FACS staining reagents is provided in Supplementary file 1 .
Facsaria iiu sorter
Manufactured by BD
Sourced in France
The FACSAria IIu sorter is a high-performance, multiparameter cell sorting instrument designed for advanced flow cytometry applications. It is capable of analyzing and sorting a wide range of cell types with precision and efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (5 mentions)
Easysep mouse cd11b positive selection kit, by STEMCELL (2 mentions)
Reliaprep rna cell miniprep system, by Promega (2 mentions)
Steponeplus real time thermocycler, by Thermo Fisher Scientific (2 mentions)
Taqman probe mixes, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Efluor 506, by Thermo Fisher Scientific (1 mentions)
Facsymphony a5, by BD (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Lsr 2, by BD (1 mentions)
Anti sca 1 pe, by BD (1 mentions)
Anti human cd44 fitc, by BD (1 mentions)
Anti human cd24 pb, by Exbio (1 mentions)
Anti human epcam percp cy5, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Accutase, by Corning (1 mentions)
Tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Revertaid reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Cystain uv precise t kit, by Sysmex (1 mentions)
15 protocols using facsaria iiu sorter
1
Comprehensive Flow Cytometry Staining Protocol
2
SSBP2 Expression in Hematopoietic Stem Cells
3
Multiparametric Flow Cytometry Profiling
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Cells were harvested with Accutase® (Corning, Catalog # 25-058-CI) and stained with anti-human CD44-FITC (BD Pharmingen), anti-human CD24-PB (Exbio) and anti-human EpCAM-APC (Biolegend) antibodies, as per manufacturer-recommended dilutions for 1 h at room temperature on a rotary shaker. Cells were analyzed in the presence of propidium iodide (1 µg/mL) using a BD LSR Fortessa (BD Biosciences). After doublet discrimination and compensation for spectral overlap, samples were analyzed using FlowJo Software v10.0.7 (BD Biosciences). For sorting, anti-human EpCAM-PerCP/Cy5.5 (Biolegend) antibody was used and cells were sorted using a BD FACS Aria IIu sorter (BD Biosciences).
4
Isolation of Murine Neutrophils
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Cells from the BM were isolated by centrifugation from the femurs and tibias of the animals, whereas spleens were mashed on 100 µm nylon cell strainer. After centrifugation, red blood cells were eliminated by treatment with ACK buffer (0.155 mM NH4Cl, 1 mM KHCO3, 0.1 mM EDTA) and filtered on a 40 µm nylon cell strainer. After ACK treatment, cells were filtered on a 40 µm nylon cell strainer. For neutrophil isolation, a first enrichment with EasySep™ Mouse CD11b Positive Selection kit (StemCell technologies, Grenoble, France) was performed followed by cell sorting of CD45+ CD11b+ Ly6G+ F4/80- cells on an ARIA IIu FACS sorter (Becton Dickinson, Le Pont-de-Claix, France).
5
Isolation and Purification of Immune Cells
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Cells from the bone marrow were isolated by centrifugation from the femurs and tibias of the animals, whereas spleens were mashed on 100 µm nylon cell strainer. After centrifugation, red blood cells were eliminated by treatment with ACK buffer (0.155 mM NH4Cl, 1 mM KHCO3, 0.1 mM EDTA) and filtered on a 40 µm nylon cell strainer. Cells from tumors of mammary glands 4 and 9 were isolated following a modified protocol from Dr J. Stingl [32 (link)]. Briefly, mammary glands or mammary tumors were minced with scalpels and digested using Tumor Dissociation Kit (Miltenyi, Paris, France) following manufacturer recommendations. After ACK treatment, cells were filtered on a 40-µm nylon cell strainer.
For neutrophil isolation, a first enrichment with EasySep™ Mouse CD11b Positive Selection kit (StemCell technologies, Grenoble, France) was performed followed by cell sorting of CD45+ CD11b+ Ly6G+ F4/80− cells (for neutrophils) and CD45+ Ly6G− F4/80+ (for macrophages) on an ARIA IIu FACS sorter (Becton Dickinson, Le Pont-de-Claix, France).
For neutrophil isolation, a first enrichment with EasySep™ Mouse CD11b Positive Selection kit (StemCell technologies, Grenoble, France) was performed followed by cell sorting of CD45+ CD11b+ Ly6G+ F4/80− cells (for neutrophils) and CD45+ Ly6G− F4/80+ (for macrophages) on an ARIA IIu FACS sorter (Becton Dickinson, Le Pont-de-Claix, France).
6
Flow Cytometry Analysis of Breast Cancer Stem Cell Markers
7
Cell Sorting and qPCR Analysis
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Cells were purified using a FACSAria IIu sorter (BD Biosciences). RNA was extracted from FACS-sorted cells by using the ReliaPrep RNA Cell Miniprep System (Promega) and cDNA synthesis was performed with the RevertAid Reverse Transcriptase kit (Thermo Fisher) as indicated by the manufacturer. qPCR analysis was performed in triplicate using TaqMan-Probe mixes (Applied Biosystems; listed in Table S5 ) with TaqMan gene expression mastermix (Applied Biosystems). qRT-PCR data were acquired on a StepOnePlus Real-Time thermocycler (Applied Biosystems) and analyzed with the StepOne Software version 2.3. Results were calculated by applying the ΔΔCT-method.
8
Cell Cycle Analysis of TSC and iXTE
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TSC and iXTE cells were grown in serum-containing medium for 5 days. After that, the cells were dissociated using 0.25% trypsin and processed using a CyStain UV Precise T kit according to the manufacturer’s protocol (Sysmex Deutschland GmbH). The DNA content was analyzed using a FACSAria IIu sorter (BD Biosciences).
9
FACS-based Cell Sorting for PDGFRα and CD40
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Cells were dissociated using 0.05% trypsin and resuspended in PBS supplemented with 3% FCS (dilution buffer). For sorting cells based on PDGFRα and CD40 expression, the cells were incubated with the respective antibodies (table S2) for 1 hour on ice and washed three times with dilution buffer to remove traces of unbound antibodies. After that, the cells were resuspended in the dilution buffer and subjected to sorting in FACSAria IIu sorter (BD Biosciences). Single viable cells were first selected on the basis of FSC and SSC gating, and either PDGFRα+ and Venus+ cells or PDGFRα+ and CD40+ cells were collected depending on the experimental setup. FlowJo software was used for data analysis and plotting.
10
RNA Extraction and qPCR Analysis from FACS-Sorted Cells
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Cells were purified using a FACS Aria IIu sorter (BD). RNA was extracted from FACS‐sorted cells by using the ReliaPrep RNA Cell Miniprep System (Promega). Residual genomic DNA was digested using DNase I (Thermo Fisher). cDNA synthesis was performed with the RevertAid Reverse Transcriptase (RT) kit (Thermo Fisher) as indicated by the manufacturer. Removal of genomic DNA was verified by PCR on RNA samples subjected to cDNA synthesis in absence of RT. Quantitative (q)PCR analysis was performed by using the TaqMan‐probe mixes (Applied Biosystems), enlisted in Appendix Table S11 , together with TaqMan‐gene expression mastermix (Applied Biosystems). qPCR data were acquired on a StepOnePlus real‐time thermocycler (Applied Biosystems) in triplicates and analyzed with the StepOne Software v2.3. Results were calculated by applying the ΔΔCT‐method.
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