At the time of the fifth injection, mice were retro-orbitally injected with 300 µl of a P. falciparum infected huRBC suspension in RPMI at a parasitemia of 1% (all the developmental forms, i.e. rings, trophozoites and schizonts were present).
NOD/SCID mice were retro-orbitally injected with 400 µl huRBC every 3 days to ensure a satisfactory proportion of huRBC (i.e. chimerism), at the time of infection (≈60%).
For testing the response of Vβ T cell clones to superantigen, mice were retro-orbitally injected with a single dose of 1 µg SEA (Staphylococcal Enterotoxin A, Sigma-Aldrich) per mouse, in 200 µl PBS.
C3H/HeN mice were retro-orbitally inoculated with a range of doses of B31-A3 (WT), Δp66, p66 cc or BDel202 208 bacteria.
At this point, mice were retro-orbitally injected with 12 μg of purified and filtered sterile HCAb1 (n = 2) and HCAb2 (n = 3) in 100 μL of sterile saline.
Erythrocyte-depleted BM cells (RBC lysing buffer from Sigma), from a pool of young CD45.1+ WT donor mice, were retro-orbitally transplanted (BMT) into 2- and 18-month-old CD45.2+ IL7R−/− host mice (2 × 10 cells/mouse).
Experiment-B: to test competence for thymic T-lymphopoiesis in LPC niches (both BM and thymic niches) of different ages, ~2 × 10 erythrocyte-depleted BM cells from a pool of young CD45.1+ WT mice were retro-orbitally injected into ~2-, 6-, 8, 12~13-, and 18-month-old non-irradiated CD45.2+ IL7R−/− host mice.
To distinguish whether the abnormal response of Foxn1Δ/Δ T cells was directly due to the developmental defect in the thymus or the environmental defect in periphery, 4×106 sorted Ly5.1 wild-type peripheral T cells per mouse were retro-orbitally transferred into Foxn1Δ/Δ adult mice.
The complete microarray dataset is MIAME compliant and the raw data has been deposited in the MIAME compliant database Gene Expression Omnibus (GEO) with the accession number GSE25343. 1 µg of total RNA from each individual mouse was retro-transcribed into cDNA by SuperScript First-strand Synthesis System (Invitrogen, Carlsbad, CA), following the manufacturer's instructions.
HIS mice were injected retro-orbitally with 200 uL of PBS containing the specified dose of virus per mouse.
On day 20 of pregnancy, the female mice were given retro-orbital injections of IL-4 or 13.
