Accuri c6 facs analyzer

To evaluate PVL and CD4⁺ T-cell decline, peripheral blood was obtained on a weekly and bimonthly basis, respectively. Plasma RNA was extracted utilizing the E.Z.N.A. Viral RNA kit (Omega bio-tek, Norcross, CA) and the manufacturer’s instructions. Viral load was quantified using the iScript One-Step RT-PCR kit with SYBR Green (BioRad, Hercules, CA) per the manufacturer’s instructions as described previously.^{10 (link),14 (link)} CD4⁺ T-cell decline was determined by staining whole blood with fluorophore conjugated anti-human CD45-FitC (eBioscience), CD3-PE (eBioscience), and CD4-PE/Cy5 (BD Pharmigen, San Jose, CA) antibodies. BD Accuri C6 FACS Analyzer was used to determine cell counts as described previously.^{13 (link),14 (link)} The CD4⁺ T-cell decline between the infected and uninfected mice was assessed using a two-tailed Student’s t-test (p<0.001).

Flow cytometry was used to assess human cell engraftment levels in humanized SIVsm infected and uninfected mice. Peripheral blood was collected in heparinized capillary tubes by tail vein puncture bimonthly. 5 ul of FcγR-block (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) was added to the blood cell pellet and then stained with fluorophore conjugated hCD45-FITC (eBioscience), hCD3-PE (eBioscience) and hCD4-PE/CY5 (BD Pharmingen, San Jose, CA) for 30 min. Erythrocytes were lysed using a RBC lysing kit (BD sciences). After lysing, stained cells were washed twice with washing buffer (BD Sciences) and fixed in 1% paraformaldehyde. Stained cells were analyzed using BD Accuri C6 FACS analyzer. In order to measure CD4⁺ T cell depletion in SIV infected mice, CD3⁺ T cell levels were calculated as a ratio of the entire CD45⁺ (lymphocyte common antigen marker) population. The CD4⁺ T cell level was then determined based on the ratio of the entire CD3⁺ T cell population‥ Engraftment levels of CD45, CD3 and CD4 were measured before infection as a control. CD4⁺ T cell decline was assessed using a two-tailed Student’s t-test (<0.05) to compare the two groups of mice, infected and uninfected. All flow data was analyzed using the FlowJo software package (FlowJo LLC, Ashland, Oregon).