BioMag Goat Anti-Rat IgG (500 ml)

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"Cell isolation and adoptive transfers
T cells were enriched by negative selection. Cell suspension was labeled using a combination of anti-B220 (RA3-6B2, Biolegend), anti-CD11b (M1/70, Biolegend), anti-Ly6G (1A8, Biolegend), and anti-DX5 (CD49b, Biolegend) antibodies followed by magnetic isolation using anti-rat BioMag beads (QIAGEN). B cells were enriched with a MagniSort mouse B cell enrichment kit (Affymetrix eBioscience). T and B cells were labeled with 10 μM of either CFSE, CMTMR for 10 min or 100uM CMAC (Invitrogen, life Technologies) for 30 min and 10 million of each were adoptively transferred intravenously. Unless otherwise specified, mice were imaged 24 h after transfer.
For the inhibition of Gi protein-coupled receptors assay, 20 millions GFP+ or CMAC labeled T cells were incubated 2 h at 37°C with 100ng per ml pertussis toxin (Quadratech Dagnostics Ltd.) or PBS, washed and 10 millions of each were co-injected into the tail vein of hCD2-DsRed mice few h before imaging.
To image the blood flow, blood from hUbi-GFP mice was collected in Alsever’s solution and washed with cold-PBS followed by a density gradient purification (Ficoll-Paque Plus). Cells at the bottom of the tubes were collected and the purity was assessed by flow cytometry based on staining with anti-CD45-PE (clone 30F11, Biolegend) and anti-TER119-PerCP-Cy5.5 (Biolegend). The purity was typically over 96%. Approximately 108 red blood cells were injected intravenously minutes before imaging.
To highlight the vasculature, 100 μg Dextran Fluorescein 2000 kDa (Invitrogen) was injected intravenously immediately before acquiring time lapse of pre-identified regions of interest."

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