C57bl 6 and fvb mice

Naive 6–8-week old male immunocompetent C57BL/6 and FVB mice (Charles River Laboratories, UK) were housed in groups of six in a pathogen-free facility with 12-h light cycles, in individually ventilated cages on woodchip bedding, with access to water and food ad libitum, at 22 °C (range 21–24 °C) and 50% humidity (range 35–75%), with environmental enrichment and bedding material, and monitored for body weight changes twice weekly.²⁶ In total, 2 × 10⁶ TRAMP-C1 (C57BL/6 mice) or 1 × 10⁶ MyC-CaP (FVB mice) cells in PBS and 1:1 high concentration phenol red-free Matrigel^® (Corning) were injected into the flank of mice under isofluorane inhalational anaesthesia on a heat mat. Tumours were measured in the home cage pre- and post treatment using digital callipers three times per week (tumour volume = π/6 × length × width × height). When tumours reached 100–120 mm³, mice were assigned to treatment groups on a first come, first allocated basis using a randomly generated treatment list (GraphPad Prism 8, USA). All mice were culled by a schedule one procedure at end point in the morning using pentobarbitone injection overdose followed by cervical dislocation according to institutional guidelines.²⁶

Male and female C57BL/6 and FVB mice between 8 and 12 weeks of age were purchased from Charles River Laboratories (Morrisville, NC). MMTV-PyMT mice were initially purchased from Jackson Laboratories and crossed seven generations onto a C57BL/6 background [18 (link), 19 (link)]. Cx3cr1^GFP/GFP mice were also purchased from Jackson Laboratories (Bar Harbor, Maine) and cross to C57BL/6 to generate Cx3cr1^wt/GFP. All mice were housed at a barrier-free and specific-pathogen-free facility at Duke University School of Medicine (Durham, NC). All procedures were approved by the Institutional Animal Care and Use Committee at Duke University.

C57BL/6 and FVB mice were purchased from Charles River Laboratories and housed (4 ~ 5 animals per cage) with a 12/12 h light/dark cycle and with ad libitum access to food and water. The housing, breeding, and animal experiments were in accordance with the approved protocol from our Institutional Animal Care and Use Committee, according to the PHS Policy on Human Care and Use of Laboratory animals.
Adult mice of both genders (~6 months old) were sacrificed by cervical dislocation, and the brains were removed immediately according to each experiment. Animals were then either transcardial perfused (see below) or their forebrains were dissected out in ice cold PBS and frozen in dry ice, and stored at −80 °C for biochemical analyses.