Carboxyfluorescein succinimidyl ester (CFSE) proliferation assay

The proliferative capacity of T cells was assessed by a CFSE dilution assay in LdCen-/- immunized mice before and after challenge with virulent L. donovani parasites. Age-matched naive mice served as negative controls for Ag-specific proliferation. Splenocytes from different groups of mice were isolated, incubated in 5μM CFSE (Molecular Probes/Invitrogen) for 10 min in RPMI 1640 without fetal calf serum (FCS), followed by 5 min of quenching in ice-cold RPMI 1640 plus 10% FCS, and subsequently washed thoroughly before plating in 96-well tissue culture plates at 2×10⁵ cells/well. Cells were cultured for 5 days at 37°C with 5% CO2 under stimulation with FTAg (80 μg/ml). Cells were harvested, washed, and blocked with anti-CD16/32 (5 μg/ml) for 20 min (4°C) and stained with anti-mouse CD3 allophycocyanin–eFluor 780, anti-mouse CD4 eFluor@450, anti-mouse CD8a eFluor@605NC (eBioscience, USA) (each with 1:200 dilution; 4°C) for 30 min. For analysis, single live cells (dead cells were excluded based on staining with the LIVE/DEAD Aqua dye) were gated for CFSE stained CD4⁺T cells and CD8⁺T cell and proliferation was calculated. Cells were acquired on an LSR II (BD Biosciences) equipped with 405-, 488-, 532-, and 638- nm laser lines using FACSDiva 6.1.2 software. Data were analyzed with FlowJo software version 9.1.5 (Tree Star, San Carlos, CA).