Cd31 pecam 1 fitc

To generate a bone tropic variant of 4T1, cells were isolated from spontaneous bone metastases: Following orthotopic injection and primary tumor excision, mice were monitored for signs of morbidity/relapse every other day. Bones were then flushed in order to isolate cancer cells which were cultured thereafter. Cells were cleaned from possible immune/endothelial cell contamination by FACS sorting for EpCAM⁺CD45⁻CD31⁻, and reinjected orthotopically for another cycle of in vivo selection. Antibodies used: CD31(PECAM-1)-FITC (eBioscience, 11-0311-82, diluted 1:50), CD45-PE/Cy7 (eBioscience, 25-0451-82, diluted 1:200), and EpCAM-APC (eBioscience, 17-5791-80, diluted 1:100). Cells were designated 4T1.1 (first cycle) and 4T-Bone (second cycle). Sorting was performed using BD FACSAria II.

Orthotopic 4T1 tumours or lungs of 4T1-tumour-bearing mice were isolated. Disassociation of tumours was performed as described²¹ . For dissociation of lung tissue, lungs were minced into small pieces and incubated for 45 min with DMEM supplemented with 0.1% collagenase II (Worthington, LS4176) and 0.1% Dispase II (Sigma-Aldrich, 4942078001). Single cell suspensions were incubated with anti-mouse CD16/CD32 (eBioscience, 16-0161-82, diluted 1:50) for 15 min to reduce non-specific staining, followed by staining with the following anti-mouse antibodies: CD31 (PECAM-1)-FITC (eBioscience,11-0311-82, diluted 1:50), CD45-PE/Cy7 (eBioscience, 25-0451-82, diluted 1:200), and EpCAM-APC (eBioscience, 17-5791-80, diluted 1:100). ECs were isolated as CD31⁺CD45⁻EpCAM⁻ cells. For myeloid cell isolation, tissues were disassociated into single cells suspensions as described above, and stained with the following anti-mouse antibodies: CD45-PE/Cy7 (eBioscience, 25-0451-82, diluted 1:200), CD11b-PerCP/Cy5.5 (eBioscience; 45-0112-82, diluted 1:100), Ly-6G-APC (Biolegend, 127614, diluted 1:200) Ly-6C-FITC (Biolegend, 128006, diluted 1:200). DAPI was used to exclude dead cells. Sorting was performed using BD FACS Aria II.