5 laser lsr 2 cytometer

BM cell suspensions from all mouse strains described above were prepared by crushing the long bones (2 femurs and 2 tibias per mouse) into DMEM/3% FCS. Bone fragments were removed by filtration through 40-μm filters. Splenocyte suspensions were obtained by mashing the organs through sieves into DMEM/3% FCS, washing by centrifugation and filtering through a 40-μm filter cap. Liver hematopoietic mononuclear cells were obtained by mashing entire livers through sieves, after which the cells were washed in DMEM/3% FCS and centrifuged using a Percoll (GE Healthcare) gradient (40% Percoll layered over 80% Percoll) for 30 mins at 2,000 rpm to remove hepatocytes and other non-hematopoietic cells. Cells localized at the interface were recovered, diluted in DMEM/3% FCS, centrifuged and filtered through 40-μm filter caps. Single-cell suspensions were stained as previously described (20 (link)). Monoclonal antibody conjugates used for flow cytometry are listed in Supplementary Table S1. Cells were analyzed on a 5-laser LSR II cytometer equipped with 355, 405, 488, 561, and 640-nm lasers (Becton Dickinson, San Jose, CA), and the data were analyzed with FlowJo V9 software (TreeStar, Ashland, OR).

Single-cell suspensions were stained with an anti-hPSMA-APC antibody (clone REA408; 2.5 μl/test; Miltenyi Biotech) for 30 min at 4 °C. Samples were fixed and permeabilized using the Cytofix/Cytoperm kit from BD and stained with an anti-phospho-γH2A.X-FITC antibody (1:800, 20 min, RT, dark; Millipore) and DAPI to analyze DNA damage. Samples were measured on a 5-laser LSRII cytometer (Beckton Dickinson) and analyzed using FlowJo software (Tri Star).