Comprehensive analysis of 12 different cytokines in the plasma samples from rhesus (n = 13) showed significantly higher plasma levels upon arrival at the new facility compared to levels prior to relocation.

<p>Comprehensive analysis of 12 different cytokines in the plasma samples from rhesus (n = 13) showed significantly higher plasma levels upon arrival at the new facility compared to levels prior to relocation.</p

IFN-γ and perforin ELISpot response to mitogens.

<p>Triplicate wells of the 96-well microtiter plates, pre-coated with IFN-γ (A) or perforin antibody (B), IFN-γ antibody with male and female (C), or perforin antibody with male and female (D) were seeded with 10⁵ PBMCs on days 0, 2, and 30 after relocation, stimulated with 1 μg of each of the mitogens for 32 h at 37°C, and then washed and stained with biotinylated secondary IFN-γ or perforin antibody. The total number of spot forming cells (SFCs) in each of the mitogen-stimulated wells was counted and adjusted to control medium as background. See the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188694#sec002" target="_blank">Materials and methods</a>” section for experiment details. P values <0.05 were considered statistically significant. <i>Symbol</i>: <i>* p<0</i>.<i>05; **p<0</i>.<i>01</i>.</p

Proliferative response of PBMCs to mitogens.

<p>PBMCs that were isolated from blood samples from days 0, 2, and 30 after relocation of the monkeys as a group (A) and from the male and female groups (B) of monkeys were used for determining proliferative response to various mitogens, using the standard MTT dye reduction assay. Proliferation responses were expressed as optical density (OD) after blank (i.e., medium only) subtraction. P values of <0.05 were considered statistically significant.</p

Cytokine bead array (CBA) analyses of plasma samples.

<p>In duplicate wells of the 96-well filter plate, 25 μL of plasma from monkeys on days 0, 2, and 30 was incubated with 25 μL of cytokine-coupled beads overnight at 4°C, followed by washing and staining with biotinylated detection antibody. The plates were read on Biorad 200 with use of Luminex technology, and the results were expressed as pg/mL concentration. The minimum detectable concentrations in pg/mL for IFN-γ (2.2), IL-2 (0.7), IL-4 (2.7), IL-6 (0.3), IL-10 (6.2), IL-13 (5.8), IL-12/23(p40) (1.2), IL-1b (1.2), IL-ra (2.4), TNF-α (2.1), MCP1 (3.1), and VEGF (13.6) were used for considering positive responses. See the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188694#sec002" target="_blank">Materials and methods</a>” section for experimental details. P values <0.05 were considered statistically significant. <i>Symbol</i>: <i>* p<0</i>.<i>05; **p<0</i>.<i>01; *** p<0</i>.<i>0001</i>.</p

Cortisol analyses of plasma samples.

<p>In duplicate wells of the 96-well filter plate, 25 μL of plasma from monkeys on days 0, 2, and 30 was incubated with 25 μL of diluted (1:50) plasma. See the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188694#sec002" target="_blank">Materials and methods</a>” section for experimental details. P values <0.05 were considered statistically significant.</p

Phenotypic analyses of lymphocytes in young, adult and aged groups of female squirrel monkeys.

<p>(<b>A</b>) Gating scheme for phenotype analyses of the different cell markers in the peripheral blood from a representative animal. The lymphocytes were first gated based on forward scatter (FCS) versus side scatter (SSC) and then CD3+ T cells and CD20+ B cells were positively identified. Further analyses of CD3+ T cells show CD4+ T cells, CD8+ T cells and CD4+CD8+ double positive T cells. The specificity of staining for the different markers is ascertained based on isotype control antibody staining used for each pair of combination markers as shown. (<b>B</b>) Numbers of different lymphocyte populations in the three groups of animals: Aliquots of EDTA whole blood were stained with fluorescence labeled antibodies to the CD3+, CD4+, CD8+, CD20+ and CD16+ lymphocytes. Values on the Y-axis are the absolute number of Lymphocytes cells. The results shown are average of two separate experiments and the standard deviation values did not exceed 15% of the mean value. P values were considered at p<0.05.</p

Comparison of Hematologic Parameters Between young, Adult and aged female.

<p>Abbreviation of tests: White Blood Cells (WBC), Red blood cells (RBC), Hemoglobin (Hgb), Hematocrit (HCT), Mean Corpuscular volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), and Red Blood Cell Distribution (RDW), Platelet Counts (PLT), Mean platelet Volume (MPV).</p

Phenotypic analyses of memory T cell subpopulations.

<p>(<b>A</b>) Gating scheme for the analyses of the different T cell subsets in the peripheral blood from a representative animal. The lymphocytes were first gated based on forward scatter (FCS) versus side scatter (SSC), and then live lymphocytes were identified based on SSC and live population (the later based on Aqua Live/Dead reagent (Invitrogen, Carlsbad, CA). The T cells were then positively identified by CD3 expression followed by the detection of the CD4+ CD8− (CD4+ T cells) and CD4− CD8+ (CD8+ T cells) populations within the CD3+ T cells. On the basis of CD28 and CD95 expression, the CD4+ and CD8+ T cells were further differentiated into naive (Tn CD28+ CD95−), central memory (Tcm CD28+ CD95+) and effector memory (Tem CD28− CD95+) subsets. The specificity of staining for the different markers is ascertained based on fluorescence minus one (FMO) controls shown and as described in the methods section. Blood samples from the three different age groups of squirrel monkeys were stained, and analysed for T cell subpopulations by flow cytometry as described in the methods section. Percentages of naïve (CD28+ CD95−), central memory (CD28+CD95+), and effector memory (CD28−CD95+) subsets of CD4 (<b>B</b>) and CD8 T cells (<b>C</b>) were compared between the three different groups. The results shown are average of 10 monkeys in each group and <i>P</i><0.05 was considered statistically significant.</p

IFN-γ ELISpot response to mitogens.

<p>Triplicate wells of the 96-well microtiter plates, pre-coated with IFN-γ antibody, were seeded with 10⁵ PBMC from the monkeys in the three different age groups studied and stimulated with 5 µg of each of the mitogens for 36 h at 37°C followed by washing and staining with biotynylated second IFN-γ antibody. The total number of spot forming cells (SFC) in each of the mitogen-stimulated wells was counted and adjusted to control medium as background. See methods section for experimental details. The results shown are average of two separate experiments and the standard deviation values did not exceed 15% of the mean value. P values were considered at p<0.05.</p

Proliferative response of PBMC to mitogens.

<p>PBMC isolated from the blood samples of the squirrel monkeys were used for determining proliferative response to different mitogens using the standard [3H] thymidine incorporation assay. The proliferation responses are expressed as delta (Δ) counts per minuets (CPM), representing increase in radioactivity incorporated in the presence of the mitogen over to that in medium control. The results shown are average of two separate experiments and the standard deviation values did not exceed 15% of the mean value. P values were considered at p<0.05.</p