Igd percp cy5

At indicated times post-infection a proportion of lymphocyte cultures representing ~200,000 cells were pelleted at 400g for 3 minutes into 96-well round bottom plates. Cells were washed once with PBS and resuspended in 200μl PBS containing (0.4ng/ml) fixable viability stain (BD Cat# 564406) and incubated at room temperature for 10 minutes. Cells were pelleted and resuspended in 100μl cold PBS without calcium and magnesium containing 5% FBS, and 0.1% Sodium Azide (FACS Block) and incubated on ice for 15 minutes after which 100μl cold PBS containing 0.5% FBS and 0.1% Sodium Azide (FACS Wash) was added. Cells were pelleted and resuspended in FACS Wash containing B cell phenotype panel as follows for 15 minutes on ice: (volumes indicated were routinely used for up to 0.5(10)^6 cells and were based on titration of the individual antibodies on primary tonsil lymphocyte specimens) Ig Lambda Light Chain-V450 (2μl), CD19-PE (8μl), CD38-PECy7 (3μl, BD Cat# 560667), IgD-PerCP Cy5.5 (2.5μl, BD Cat# 561315), CD138-APC (4μl, BD Cat# 347207), CD27-APC H7 (2.5μl BD Cat# 560222), Ig Kappa Light Chain-Alexa700 (2μl). After incubation, 100μl FACS Wash was added and pelleted lymphocytes were washed with a further 200μl of FACS Wash prior to being resuspended in 200μl FACS Wash for analysis. Data was acquired on a BD LSR2 Flow Cytometer and analyzed using FlowJo software.