Allophycocyanin apc cd11b

Splenocytes were resuspended at room temperature in PBS at 5.00 × 10⁶ cells/mL and divided into 100μl samples for fluorescent labeling using standardized protocols for flow cytometry. Cells were labeled with Aqua live-dead viability label for 20 minutes at room temperature. Following viability labeling, cells were centrifuged at 500 × g for 10 minutes and washed in 200μL staining buffer. Cells were then resuspended in 100μl of staining buffer and incubated with 25μg/mL Fc Block™ for 10 minutes. Cells were then cooled to 4°C by refrigeration and either left unlabeled, labeled with fluorescein isothiocyanate (FITC)-CD45.1, Alexaflour700 (Alexa-700)-CD45.2, phycoerythrin (PE)-CD3, or allophycocyanin (APC)-CD11b, (eBiosciences, San Diego, CA), or an equal concentration of their fluorescently conjugated corresponding isotype controls for 30 minutes at 4°C. The two strains differ in CD45 alleles, with BTBR expressing the CD45.1 allele and C57 expressing the CD45.2 allele, enabling clear distinction between cell populations following transplantation. Following labeling, cells were washed twice with 200μL staining buffer, resuspended in 300μL staining buffer and analyzed by flow cytometric analysis using a BD-LSRII (BD Biosciences). Flow cytometry data was analyzed with FlowJo software.