Leukocyte isolation from blood and tissues and staining for flow cytometry was conducted as described (18 (link)). Briefly, leukocytes from blood and tissues were stained with anti-VLRA rabbit polyclonal serum (R110), anti-VLRB mouse monoclonal antibody (4C4) and biotinylated anti-VLRC mouse monoclonal antibodies (3A5) and with matched secondary reagents. Staining and washes were in 0.67× PBS with 1% BSA. Flow cytometric analysis was performed on an Accuri C6 flow cytometer (BD Biosciences). VLRA+, VLRB+, VLRC+ and VLR triple-negative (TN) cells in the lymphocyte gate were sorted on BD FACS Aria II (BD Bioscience) for quantitative real-time PCR analysis. The purity of the sorted cells was > 95%. Lampreys were injected intra-coelomically with 25 μg phytohaemagglutinin (PHA)-L (Sigma) as described previously (17 (link), 18 (link)).
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Leukocyte Isolation and Staining for Flow Cytometry
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Variable analysis
independent variables
- Injection of lampreys with 25 μg phytohaemagglutinin (PHA)-L
- Proportion of VLRA+, VLRB+, VLRC+ and VLR triple-negative (TN) cells in the lymphocyte gate
- Quantitative real-time PCR analysis of the sorted cells
- Leukocyte isolation from blood and tissues
- Staining leukocytes with anti-VLRA rabbit polyclonal serum (R110), anti-VLRB mouse monoclonal antibody (4C4) and biotinylated anti-VLRC mouse monoclonal antibodies (3A5) and with matched secondary reagents
- Staining and washes in 0.67× PBS with 1% BSA
- Flow cytometric analysis on an Accuri C6 flow cytometer
- Cell sorting on BD FACS Aria II with > 95% purity
- None specified
- None specified
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