Prepared single cell suspensions of mouse tissues and in vitro treated cancer cells were incubated with mouse FcR Blocking Reagent (Miltenyi) followed by incubation with (a combination) of specific pre-labelled antibodies or in combination with fluorescently-labelled secondary antibodies (Invitrogen) (see Supplementary Information ). Dead cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI; both Sigma). The LSRFortessa cell analyser running FACSDiva software (BD Biosciences) and FlowJo software was used. Tumour cells were flow-sorted using the Influx cell sorter running FACS Sortware sorter software (BD Biosciences). MMTV-PyMT cells were used in experiments immediately after sorting and sorted 4T1 cells cultured in adherent conditions for 3 days prior to western blot analysis.
Facs sortware sorter software
Manufactured by BD
The FACS Sortware sorter software is a core component of flow cytometry systems. It provides the essential functionality to control and manage the sorting of cells or particles based on their physical and fluorescent characteristics. The software enables users to configure, operate, and analyze the sorting processes within a flow cytometry workflow.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa cell analyser, by BD (2 mentions)
Mouse fcr blocking reagent, by Miltenyi Biotec (2 mentions)
Fluorescently labelled secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facsdiva software, by BD (1 mentions)
2 protocols using facs sortware sorter software
1
Tumor Cell Immunophenotyping and Sorting
2
Intracellular Staining of Macrophages
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For intracellular staining of macrophages for vimentin, tissue was digested as described to prepare a single-cell suspension. The preparation was incubated with LIVE/DEAD stain Zombie Violet before being incubation with FcR Blocking Reagent followed by extracellular staining with CD11b-FITC and Ly6G-PE. The preparations were then fixed and permeabised before staining with Vimentin-APC. The LSRFortessa cell analyser running FACSDiva software (BD Biosciences) and FlowJo software was used. Prepared single-cell suspensions of PyMT tumour preparations were incubated with mouse FcR Blocking Reagent (Miltenyi) followed by incubation with CD24-e450, CD45-PE, CD31-PE, Ter119-PE, AXL-biotin (in combination with Streptavidin-APC780). Dead cells were stained with or propidium iodide (PI; Sigma). Tumour cells were flow-sorted using the Influx cell sorter running FACS Sortware sorter software (BD Biosciences).
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