"Single-cell suspensions of splenocytes were analyzed by flow cytometry to quantify various cell populations, as previously

described (White et al. 2005; Auttachoat et al. 2009). The populations evaluated were: B-lymphocytes (B-cells, CD45þ rats, Igþ mice), total T-lymphocytes (T-cells, CD5þ rats, CD3þ mice), Tcell subsets (CD4þCD5þ and CD8þCD5þ rats, CD4þCD8, and CD4-CD8þ mice), NK cells (NKþCD8þ rats, NK1.1þCD3 mice), and macrophages (His36þ rats, Mac-3þ mice). Isotypematched irrelevant antibodies were used as controls. Cells were counted on a Becton Dickinson FACScan Flow Cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA). Nonviable cells and red blood cells (RBC) were eliminated using a gate-setting that excluded propidium iodide (PI) fluorescence and a forward scatter threshold above RBC size. For each sample, 5000 PI-negative events were counted. All data were analyzed with CellQuest software v. 3.2.1 (Becton Dickinson, San Jose, CA)."

Statistical analyses: "All results, except the host resistance data, are presented as mean ± SE for 7–8 animals per group. All statistical analyses were conducted using SAS software (SAS Institute, Cary, NC). Jonckheere’s Test was used to test for dose-related trends. Data which typically exhibit a normal distribution were analyzed using a parametric multiple comparison procedure. If a significant trend was observed Williams’ test was used; if the trend was not significant Dunnett’s test was used. Positive control and vehicle control data were compared by Student’s t-test. Data for other endpoints were analyzed using a non-parametric multiple comparison procedure. If a significant trend was observed Shirley’s test was used; if the trend was not significant Dunn’s test was used. Positive control and vehicle control data were compared by a Wilcoxon Rank Sum Test. Host resistance data were analyzed by a Cochran–Armitage test, to establish single trend p values on binary moribundity data, and by a Fisher’s exact test to determine pairwise significance. Data that were different from control at p>= 0.05 were considered statistically significant.

Female B6C3F1/N Mice