Liberase enzyme

After injection of oncogenic cells, the flies were incubated at 25 °C for 11 d to let the injected cells grow inside the fly body. While the first isolated cell suspension was kept on ice, the remaining tissues were further homogenized in the dissociation buffer with 0.5 mg/mL liberase enzyme (Sigma) at room temperature for 15 min. The dissociated tissues were repeatedly washed by PBS, and the remaining cells were harvested. All collected cells were stained with DAPI to discriminate dead cells and applied on a BD FACS Arria II cell sorter. The sorted GFP+DAPI cells (labeled as in vivo D11 OC) and GFP-DAPI cells (labeled as Host) were immediately dissolved in TRIzol (Invitrogen) for the subsequent total RNA isolation and RNAseq analysis. Embryos from at least 500 transgenic flies (Bloomington: BL1691) were collected, bleached (3 to 5 min to remove chorion membranes) and washed with phosphate-buffered saline (PBS), then placed within a cell strainer (40 μm, Falcon) and homogenized within the PBS solution using the white end of the plunger (sometimes inside the protective cap) of an insulin injector. The embryonic cell suspension was then purified using Ficoll Paque Plus solution (GE Healthcare). After centrifugation at 400 g for 30 min, the alive cells located in the interphase were collected and resuspended in PBS.

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 assay^{27 (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.