Six to eight-week-old female hamsters were immunized with 106 total stationary-phase LmexCen−/− parasites by intradermal injection in the left ear in 10 μl PBS using a 29-gauge needle (BD Ultra-Fine). In addition, the age-matched control group of animals were infected with 106 total stationary-phase L. mexicana wild-type (LmexWT) promastigotes. Lesion size was monitored weekly by measuring the diameter of the ear lesion using a direct reading vernier calliper. Parasite burdens in the ear, draining lymph node (dLN), spleen, liver and bone marrow tissues were determined by limiting dilution assay27 (link). Ear tissues were treated with Liberase enzyme (Sigma-Aldrich) (0.15 mg/ml) in DMEM mediun at 37 °C. After 90 min of incubation tissues were grinded to make single cell suspensison using BD medimachine system. Cells were washed two times with M199 medium. Lymph nodes, spleen, liver and bone marrow were harvested and homogenized with a cell strainer in 2 ml of M199 medium supplemented with 10% FBS and 1% Penicillin/Streptomycin. To determine parasite burden cell suspension from each organs were serially diluted in 96 well tissue culture plates. After 12 days of incubation at 26 °C, plates were examined with a inverted microscope and parasite burden were calculated for each organ.
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