Nod scid il2yr female mice

For tail vein injection assays, 6–8-week-old NOD/SCID/IL2yR−/− female mice were supplied by Jackson Labs (Cat #005557).

The experiments were conducted following protocols approved by ISMMS Institutional Animal Care and Use Committee (IACUC) (protocol number LA11-00122). 6–8-week-old NOD/SCID/IL2yR−/− female mice (Jackson Labs, Cat# 005557) were used. Cells were harvested with 2mM EDTA-PBS, resuspended in 100 μL HBSS (SKmel147: 1.25 × 10⁵ cells; 4L: 5 × 10⁴ cells), and injected into one of the lateral tail veins with a 27-gauge needle. At the experimental endpoint (SKmel147: 3 weeks; 4L: 5 weeks), mice were euthanized by cervical dislocation, lungs were cleared of blood by right ventricle PBS perfusion, and imaged ex vivo for GFP and mCherry fluorescence on a Cytation 7 instrument (BioTek). Quantification of lung colonies was performed using Gen 5 software (BioTek). Harvested lungs were fixed in neutral buffered formalin for 24 h, paraffin embedded and sectioned at 5 μM thickness. Sections were obtained from three levels spaced 100 μM, stained with H&E and acquired at 20× magnification on a NanoZoomer S210 slide scanner (Hamamatsu). Metastatic colonies were identified morphologically and counted using NDP.view 2 software (Hamamatsu). Lung area on each section was calculated by NDP.view 2 software by outlining the edges of the tissue. Slides were blinded to the reviewer (C.N.) prior to analysis.