T cells were purified from the spleens or draining lymph nodes of IRBP1–20‐immunized mice by positive selection using a combination of FITC‐conjugated anti‐CD3 antibody and anti‐FITC antibody‐coated Microbeads, followed by separation using an autoMACS separator system according to the manufacturer's suggested protocol (Miltenyi Biotec, Auburn, CA). The purity of the isolated cells, determined by flow cytometric analysis using PE‐conjugated antibodies against αβ or γδ T cells, was >95%.
Automacs separator system
Manufactured by Miltenyi Biotec
Sourced in Germany
The AutoMACS Separator system is a magnetic cell separation instrument that enables the isolation of target cells from heterogeneous cell populations. The system utilizes magnetic bead-labeled cells and a magnetic field to capture and separate the desired cell type. The core function of the AutoMACS Separator is to provide a reproducible and automated cell separation process.
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5 protocols using automacs separator system
1
Purification of CD3+ T Cells
2
Isolation of αβ and γδ T Cells
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For examination of cell function, the immunized mice were euthanized 13 days after immunization by exposure with an overdose of sodium pentobarbital. αβ T cells were purified from the spleens or draining lymph nodes of IRBP1-20-immunized TCR-δ-/- mice and γδ T cells from immunized B6 mice, by positive selection using a combination of FITC-conjugated anti-CD3 antibody and anti-FITC antibody-coated Microbeads, followed by separation using an auto-MACS separator system according to the manufacturer’s suggested protocol (Miltenyi Biotec, Auburn, CA). The purity of the isolated cells, determined by flow cytometric analysis using PE-conjugated antibodies against αβ or γδ T cells, was >95%.
3
Scratch and Transwell Assays for Macrophage and Monocyte Migration
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For random migration scratch assays with human MΦ in six-well plates, cells were treated with MCF-7 control or decoy VCM/ACM for 30 min and washed three times with PBS before fresh MΦ media was added. Scratches were applied with a small pipette tip in a marked area and scratches were acquired using Canon EOS 600D camera (Canon) and a transmitted-light microscope (AxioVert 40; Zeiss, Oberkochen, Germany). The cell-free area within the scratch was calculated using ImageJ software. For migration assays of human monocytes toward MCF-7 control or decoy VCM/ACM, primary human monocytes were isolated from human blood PBMCs by using CD14 microbeads and the AutoMACS Separator system (Miltenyi Biotec). Cells were washed with PBS and 2 × 106 cells were added onto Transwell inserts (6.5 mm Transwell with 5.0 µm pores; Corning, NY, USA). Monocytes were allowed to migrate for 2 h. Cells were collected from the lower chamber and the number of cells was determined using Neubauer counting chamber (Labor Optic, Friedrichsdorf, Germany).
4
3D Tumor Spheroid Co-culture
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3D tumor spheroids from MCF-7 cells were generated by using the liquid-overlay technique as described 18 (link). For this, 5 × 103 cells per well were seeded onto non-adherent 1% agarose-coated 96-well plates and allowed to form spheroids for 4 days. Primary human monocytes were isolated from human blood PBMCs by using CD14 MicroBeads (130-050-201; positive selection) and the AutoMACS Separator system (both from Miltenyi Biotec, Gladbach, Germany). 1 × 105 monocytes were added per spheroid and cocultures were maintained for 3 days to allow monocyte infiltration and differentiation to MΦ.
5
3D Tumor Spheroid Formation and Coculture
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To generate 3D tumor spheroids from MCF-7 and T47D cells, the liquid-overlay technique was used as described76 (link). Briefly, 5 × 103 cells per well were seeded onto non-adherent 1% agarose-coated 96-well plates and allowed to form spheroids for 4 days. To initiate spheroids from MDA-MB-231 and MDA-MB-468 cells, 5 × 103 cells were seeded per well onto 96-well round-bottom plate and centrifuged for 15 min at 1000 × g. Five percent Matrigel (Corning, NY, USA) in culture media was added to a final concentration of 2.5% per well and cells were allowed to form spheroids for 4 days. Primary human monocytes were isolated from human blood PBMCs by using CD14 microbeads (Miltenyi Biotec, Gladbach, Germany) and the AutoMACS Separator system (Miltenyi Biotec). Monocytes (1 × 105) were added per spheroid and cocultures were maintained for 3 days to allow monocyte infiltration. For light-sheet fluorescence microscopy of tumor spheroids, CD14+ cells were stained with cell dye eFluor670 (eBioscience, Frankfurt, Germany) according to the manufacturer’s protocol before coculture. To determine spheroid diameters, pictures of spheroids were taken using a Canon EOS 600D camera (Canon, Krefeld, Germany) and a transmitted-light microscope (AxioVert 40; Zeiss, Oberkochen, Germany) at × 5 magnification. Pictures were analyzed using ImageJ software.
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