Hcd8 percp cy5

Peripheral blood and spleen cell suspensions were collected weekly or at the end of study, respectively, for flow cytometry analysis. Cells were stained using the following antibodies (1:50 dilution): mCD45-AF700, hCD45-FITC, hCD3-BUV805, hCD4-BUV395, hCD8-PerCP-Cy5.5, hCD56-BV650, HLA-DR-APC, hCD38-PE, hCD57-PE-CF594 and Fixable Viability Stain 510 (catalog nos. 560510, 555482, 612895, 563550, 565310, 564057, 340691, 555460, 562488 and 564406, respectively; BD Biosciences). Data were collected on a FACSymphony flow cytometer (BD Biosciences).

The hIL-15^TgNSG mice were generated as previously described^{56 (link)–58 (link)}. All mice experiments were approved by the Wistar Institute Animal Care and Research Committee (protocol no. 201360). All animals recruited in the present study were housed in the Wistar Institute humanized mice holding room with a 12-h light:dark cycle at temperatures of 20–23 °C and 40–60% humidity. Briefly, 6- to 8-week-old female NSG-Tg(hIL-15) (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl Tg(IL15)1Sz/SzJ; Jackson Laboratory^{59 (link)}) mice were pretreated with busulfan at 30 mg kg⁻¹ and were then implanted with human fetal thymic tissue fragments and fetal liver tissue fragments under the murine renal capsule. After the surgery, mice were injected via the tail vein with CD34⁺ hematopoietic stem cells isolated from human fetal liver tissues. Human fetal liver and thymus tissues were procured from Advanced Bioscience Resources. Then 12 weeks postsurgery, human immune cell reconstitution in peripheral blood was determined using a FACSymphony flow cytometer (BD Biosciences) using the following antibodies (1:50 dilution): mCD45-AF700, hCD45-FITC, hCD3-BUV805, hCD4-BUV395, hCD8-PerCP-Cy5.5, hCD56-BV650 and Fixable Viability Stain 510 (catalog nos. 560510, 555482, 612895, 563550, 565310, 564057 and 564406, respectively; BD Biosciences). Data were analyzed with FlowJo.