Freshly dissociated tumor cell suspensions were stained with anti-F4/80 UltraPureMicroBeads (Miltenyi #130-110-443) and passed through an Automacs (MiltenyiBiotec) following manufacturer’s instructions to purify F4/80+ myeloid cells. The negative fraction was subsequently stained with anti-CD8a MicroBeads (Ly2; Miltenyi #130-117-044) and then CD8+ tumor-infiltrating T cells were purified with the Automacs. Purified CD8+ and F4/80+ cell suspensions were >90% pure. Then, purified myeloid and CD8+ T cells (2×105) were added to a monolayer of TC1-GFP cells (2×104), and co-cultured for 24 hours at 37°C before acquisition of images with an inverted microscope (TE2000-E; Nikon) equipped with a ×20 objective, and Metamorph imaging software. The fraction of the surface covered by GFP was calculated using ImageJ.
Anti f4 80 ultrapuremicrobeads
Manufactured by Miltenyi Biotec
Sourced in Australia
Anti-F4/80 UltraPure MicroBeads are a product used for the isolation and enrichment of murine macrophages expressing the F4/80 antigen. These magnetic microbeads are designed to specifically bind to the F4/80 receptor, allowing for the efficient separation and purification of macrophages from a heterogeneous cell population.
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Lab products found in correlation
2 protocols using anti f4 80 ultrapuremicrobeads
1
Quantifying Tumor Cell Cytotoxicity
2
Isolation and Purification of Synovial Macrophages
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Synoviocytes were isolated from four mice per group by digestion with type 1 collagenase as described above. Synovial macrophages were purified using anti-F4/80 magnetic beads. Synoviocytes were suspended in 90 µl PBS per 107 total cells containing anti-F4/80 UltraPure microbeads (Miltenyi Biotec, Macquarie Park, NSW, Australia) and then incubated for 30 min at 4 °C in the dark according to the manufacturer’s instruction. The F4/80 magnetically labelled cells were purified by magnetic activated cell sorting (MACS, MASC Separation columns MS; Miltenyi Biotec, Macquarie Park, NSW, Australia). The collected F4/80-positive cells were centrifuged at 300 g for 10 min. The supernatants were removed and cell pellets were then used directly for RNA isolation. Total RNA from harvested macrophage-like synoviocytes were extracted using RNeasy Mini kit (Qiagen, Doncaster, VIC, Australia) according to the manufacturer’s instructions.
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