Hcd4 pe cy5

Peripheral blood was collected bi-monthly from infected and control mice by tail vein puncture. Human cell engraftment levels were assessed by flow cytometry. 5 μl of FcγR-block (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States) was added to the blood for 5 min. The blood was then stained with fluorophore conjugated hCD45-FITC, hCD3-PE and hCD4-PE/Cy5 (BD Pharmingen, San Jose, CA, United States) for 30 min. Erythrocytes were lysed using the Whole Blood Erythrocyte Lysing kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, United States). The stained cells were then fixed in 1% paraformaldehyde and 0.45 μm-filtered. To assess CD4⁺ T cell depletion in uninfected and infected mice, the CD3⁺ T cells levels were calculated as a ratio of the entire CD45⁺ (lymphocyte common antigen) population. The CD4⁺ T cell population levels were then determined as a percentage of the entire CD3⁺ T cell population. Baseline levels of the CD45⁺, CD3⁺ and CD4⁺ cells were measured prior to infection as a control. All flow cytometry data was analyzed using the FlowJo v10.0.7 software package (FlowJo LLC, Ashland, OR, United States). CD4⁺ T cell decline was assessed utilizing a two-tailed Student’s t-test (p < 0.001) to compare the two groups of mice, infected and uninfected.