Rat anti mouse ly6g percp cy5

Seven days or 14 days following splenic injection of the breast cancer cells, CD11b⁺/Ly-6G⁺ cells were isolated from the metastasis-bearing livers. Livers were first perfused with perfusion medium (Gibco, Waltham, MA, USA, cat#17701-038) for 10 minutes at 5 ml/min. Following perfusion, livers were minced and dissociated for 1 h in a mix of collagenase A, collagenase B and hyaluronidase (2 mg/ml each, Roche, Basel, Switzerland, cat#11088793001 and 11088031001; Sigma-Aldrich, St Louis, MO, USA, cat#H3884).
To purify CD11b⁺/Ly-6G⁺ cells [34 (link),35 (link)], cells were blocked in 2.4G2 (1:10 dilution) for 30 minutes and then incubated for 30 minutes, in the dark, with an antibody cocktail containing anti-mouse CD11b-APC (1:10000 dilution, eBioscience, clone M1/70, cat#17-0112-82) and rat anti-mouse Ly-6G-PerCP-Cy5.5 (1:1000 dilution, BD Biosciences, San Jose, CA, USA, clone 1A8, cat#560602). Subsequently, cells were washed once with PBS and then incubated with Live/Dead stain (1:1000 dilution) for 30 minutes. Cells were then washed once with PBS and resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS complemented with 2% fetal bovine serum (FBS)). Samples were read on the BD FACS Canto (BD Biosciences).