Dynabeads™ Untouched™ Human Monocytes Kit

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Human neutrophils and monocytes were isolated from venous blood from a healthy female donor. Peripheral blood mononuclear cells were obtained by gradient separation with Ficoll-Paque

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Human neutrophils and monocytes were isolated from venous blood from a healthy female donor. Peripheral blood mononuclear cells were obtained by gradient separation with Ficoll-Paque

Publication protocol

"MCF7 cells were treated with E2 1 nmol/L 48 hours before experiments and labeled with Fast DiI oil red dye (Thermo Fisher Scientific cat. #D3899), 4 μg/mL in PBS, 24 hours before injections.

Human neutrophils and monocytes were isolated from venous blood from a healthy female donor. Peripheral blood mononuclear cells were obtained by gradient separation with Ficoll-Paque (GE Healthcare cat. #17-1440-02) and monocytes were separated by negative isolation by using the Dynabeads Untouched Human Monocytes kit (Thermo Fisher Scientific cat. #11350D) by following the provider's instructions. Neutrophils were isolated as described previously (PMID:30105032).

The animal ethics committee at Linköping University approved all zebrafish experiments. Transgenic Tg(fli1:EGFP)y1 zebrafish embryos were collected and maintained in E3 embryo medium with 0.2 mmol/L 1-phenyl-2-thiourea (PTU) at 28°C. Dil-labeled MCF7 cells were injected with 50% neutrophils or with 10% monocytes into the perivitelline space of 2-day-old zebrafish embryos. Correctly injected embryos were selected under fluorescence and incubated in E3/PTU + E2 1 nmol/L ± tamoxifen 1 μmol/L ± fulvestrant 1 μmol/L at 28°C during 1 or 3 days where indicated. After incubation, anesthetized zebrafish embryos were assessed for cancer cell dissemination in the tail region under fluorescence. Images of disseminated cancer cells were acquired with the Olympus CellSens Imaging software version 1.16 (Olympus CellSens Software, RRID:SCR_016238) by using an Olympus BX43 light/fluorescence microscope (10×/0.30 magnification) with excitation filters BP460-495 and BP530-550, and Olympus DP72 CCD camera.

Immunohistochemistry
Formalin-fixed tumors were paraffin-embedded and cut in 4-μm sections, deparaffinized, and exposed to rat anti-mouse F4/80 (Abcam cat. #ab6640, RRID:AB_1140040), rat anti-mouse Ly6G (BD Biosciences cat. #551459, RRID:AB_394206), rat on mouse HRP Polymer Kit (BioCare Medical cat. #RT517), rabbit anti-human von Willebrand factor (Agilent cat. #A0082, RRID:AB_2315602), mouse anti-human Ki67 (Agilent cat. #M7240, RRID:AB_2142367), rabbit anti-human N-cadherin (clone EPR19654, Abcam cat. #ab207608), mouse anti-human E-cadherin (Novus Biologicals cat. #NBP2-47827), mouse anti-human CD68 (Agilent cat. #GA60961-2), rabbit anti-human Slug (Abcam cat. #ab27568, RRID:AB_777968), rabbit anti-human Snail (Novus Biologicals cat. #NBP2–27293), rabbit anti-mouse Fibroblast activation protein (FAP; Abcam cat. #ab218164), mouse anti-human estrogen receptor α (Agilent cat. #M7047, RRID:AB_2101946), rabbit anti-human vimentin (Abcam cat. #ab16700, RRID:AB_443435), Dako EnVision + System-HRP Labeled Polymer anti-rabbit (Dako cat. #K4002), and anti-mouse (Dako cat. #K4000). Sections were counterstained with Mayer's hematoxylin. Negative controls did not show staining. Images of 10 areas of each tumor section from 3 to 4 mice in each treatment group, and three tumor sections and three normal breast tissue sections from each patient were acquired on an Olympus BX43 microscope (× 40/0.75 magnification) and digitally analyzed and quantified using ImageJ software version 1.52n (ImageJ, RRID:SCR_003070).

For immunofluorescence, sections were exposed to rat anti-mouse Ly6G, incubated with conjugated donkey anti-rat antibody (Alexa flour 594, Thermo Fisher Scientific cat. #A-21209, RRID:AB_2535795) and mounted using SlowFade Gold containing 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen cat. #S36938). Samples were visualized with an Olympus BX43 light/fluorescence microscope (× 40/0.75 magnification) with excitation filters BP360-370 and BP530-550 using an Olympus DP72 CCD camera, analyzed by CellSens Imaging software, and converted to RGB images with the same threshold using ImageJ."

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