40 μm mesh cell strainer

In the advanced (20 w.p.i.) chronic phase, spleens from NI, PI, R8 and R12 rats were extracted, and cells were obtained using a 40-μm mesh cell strainer (Becton Dickinson Labware). After red blood cells were lysed with water for 5 s, the cells were washed twice in Dulbecco’s modified Eagle medium (DMEM) and resuspended in DMEM 10% fetal bovine serum (FBS). Cells were plated in 96-well plates (2–105 cells/200 μL) containing 5 μg/mL of concanavalin A (ConA) and 1 μg/ml of lipopolysaccharide (LPS), as indicated. Proliferation was measured by incorporating 1 μCi [³H]thymidine (Amersham Pharmacia Biotech) per well during the last 24 h of a 3-day culture. Cells were then harvested onto a glass-fiber filter, using a Cell Harvester (Skatron Instruments), and radioactivity was estimated in counts per minute (cpm). Results are expressed as fold proliferation (= cpm_infected/cpm_non-infected).

The progeny from a Tg(c-myb:EGFP/nol9^+/sa1022 x nol9^+/sa1022 cross at 96 hpf was screened for the presence of EGFP and subjected to immunohistochemical staining with anti-GFP primary antibody (Invitrogen, 1:200) and anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, 1:200). Afterwards, each larva was dissected: the CHT was stored and the rest of the body was used for genotyping for the nol9^sa1022 allele. CHTs from 8 larvae of the same genotype were pooled, incubated for 30 min with 10 mM DTT (Life Technologies) in Danieau’s solution, followed by incubation with 50 μg/ml liberase (Roche) in PBS for 3 h at 37°C. Single-cell suspension was prepared by passing the solution through a 40 μm mesh cell strainer (Becton Dickinson). Flow cytometry was performed on a BD LSR-Fortessa analyzer (Becton Dickinson). Non-transgenic siblings subjected to immunohistochemical staining were used as a negative control.