Apc conjugated anti mouse cd45 antibody

Single mononuclear cell suspensions were stained with antibodies for 30 min at 4 °C in the dark. Cells were then washed with fluorescence-activated cell sorting (FACS) buffer (PBS, 0.5% FBS, 0.05% NaN₃) and resuspended in FACS buffer containing propidium iodide (PI) before being subjected to multicolor flow cytometric analysis by a FACS Canto machine (BD Biosciences). The data were analyzed using FACS Diva software (ver. 6.1.3; BD Biosciences).
The antibodies used were as follows: Alexa Fluor® 488-conjugated anti-human natural killer group 2 membrane C (NKG2C) from R&D Systems (Minneapolis, MN, USA) and anti-human CD159a (NKG2A)-PE and anti-human NKp80-PE (Beckman Coulter, Miami, FL, USA). The following antibodies against human antigens were from BioLegend: fluorescein isothiocyanate (FITC)-conjugated anti-CD8a, anti-CD16, anti-CD158a/h, and anti-CD158e1; phycoerythrin (PE)-conjugated anti-CD56, anti-NKp30, anti-NKp44, anti-NKp46, anti-CD57, anti-NKG2D, anti-CD158b, and anti-CD158f; PE-Cy7-conjugated anti-CD3 and anti-CD56; APC-conjugated anti-CD16 and anti-CD94 monoclonal antibodies; and APC-Cy7-conjugated anti-CD45. The APC conjugated anti-mouse CD45 antibody was from BioLegend.
The absolute number of cells was calculated using fluorescent beads (Flow-Count; Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions.

Approximately 300 µl of blood was collected in EDTA-coated tubes from the transplanted mice by facial venous puncture. The cells were prepared according to a previously published method^{14 (link)}. The cells were stained with antigen-presenting cell (APC)-conjugated anti-mouse CD 45 antibody (BioLegend, USA) for 30 min, washed, and suspended in phosphate-buffered saline (PBS) for analysis with a Gallios flow cytometer (Beckman coulter, USA). iRFP fluorescence was detected by the FL7 (725/20) channel. Chimerism was determined by the percentage of cells that were positive for both iRFP and CD 45.

CD31⁺ cells were stained with 2 μg ml^–1 PE-conjugated anti-mouse CD31 antibody (Clone MEC13.3; BD Pharmingen) and 2 μg ml^–1 APC-conjugated anti-mouse CD45 antibody (Clone 30F11; BioLegend) and analysed on a LSR II flow cytometer (BD Bioscience). Flow cytometric data were analysed with FlowJo software (TreeStar).