Apc cy7 conjugated anti human cd45

Single cell suspensions prepared from peripheral blood, spleen of humanized mice was stained with surface markers and analyzed on a CyAn ADP (Dako). FITC-conjugated anti-human CD40, CD24, PE-conjugated anti-human CD303, CD38, PE/Cy5-conjugated anti-human CD86, IgG, PE/Cy7-conjugated anti-human HLA-DR, PB-conjugated anti-human CD4, IgM, APC-conjugated anti-human CD10 and APC/Cy7-conjugated anti-human CD45 were purchased from Biolegend. Pacific orange-conjugated anti-mouse CD45, PE/Texas red-conjugated anti-human CD19 and LIVE/DEAD Fixable Aqua (LD7) Dead Cell Stain Kit were purchased from Invitrogen. Data were analyzed using Summit4.3 software (Dako).

Two milliliters of peripheral blood from healthy people or patients were treated with red blood cells lysis buffer. A total of 1 × 10⁶ cells were collected and incubated in 100 μL of PBS containing Zombie Yellow Dye (Biolegend, #423104) for 15 min to label live cells. After washing, samples were stained with APC/Cy7-conjugated anti-human CD45 (Biolegend, #304014), Alexa Fluor 700-conjugated anti-human CD3 (Biolegend, #344822), FITC-conjugated anti-human CD4 (Biolgend, #300506), Brilliant Violet 421-conjugated anti-human CXCR3 (Biolgend, #353716) and APC-conjugated anti-human CCR6 (Biolgend, #353416) for 30 min. Stained cells were washed twice with PBS. Cells were acquired using FACS Fortessa X-20 flow cytometer (BD Biosciences, San Jose, CA). Live CD45⁺CD3⁺CD4⁺CXCR3⁺CCR6^- cells were gated as Th1 subsets as previously described.^{59 (link)} Data analysis were performed with flowjo software (version 10.5, LLC)