Anti ccr7 pe cf594

A blood sample of 200 μl was used for CD4⁺ memory T-cell phenotyping with the following antibodies: anti-CD8-FITC (1/10, clone RPA-T8), anti-PD1-FITC (1/5, clone MIH4), anti-CD122-PE (1/10, clone Mik-B3), anti-CD62L-V450 (1/10, clone DREG-56), anti-CD4-V500 (1/20, clone RPA-T4), anti-CD95-APC (1/10, clone DX2), anti-CD45RA-PE-Cy7 (1/20, clone HI100), anti-CD45RO-PerCPCy5.5 (1/10, clone UCHL1), anti-CCR7-PE-CF594 (1/10, clone 150503), anti-CXCR3-Alexa 700 (1/10, clone 1C6/CXCR3), anti-CD27-APC-H7 (1/10, clone M-T271) (all from BD Biosciences), and anti-CD3-eFluor 650NC (1/10, clone OKT3, eBioscience). After staining, the blood sample was fixed (fix/lyse solution, BD Biosciences) and cells were acquired on a BD LSR Fortessa cytometer (BD Biosciences). Data were analysed with Flow Jo software.

PBMCs thawed and cultured overnight in IMDM (Lonza, Basel, Switzerland), supplemented with 10% human serum (HS, Sigma-Aldrich) were labelled with anti-CD8-Alexa Fluor700 (Clone: RPA-T8, BD Biosciences, New Jersey, USA), anti-CD3-PE-Cy7 (Clone: UCHT1), anti-CD45RA-PerCP Cy5.5 (Clone: HI100), anti-CD27-BV605 (Clone: 0323), anti-CD28-APC(Clone: CD28.2), anti-PD-1-BV421(Clone: EH12.2H7) from Biolegend (San Diego, USA), anti-CCR7-PECF594 (Clone: 150603), anti-CD57-FITC (Clone:NK-1, BD Biosciences) along with AlexaFluor750 labeled live/Dead stain (Life Technologies, Carlsbad, USA). Live/dead and CCR7 staining were performed at 37°C for 15 mins followed by staining with the rest of the antibodies at 4°C for 30 mins. Subsequently, cells were washed, resuspended in PBS and acquired on BD^™ LSR II (BD Biosciences, San Jose, USA). The phenotypic analysis was performed on FlowJo version 7.6.5 (Treestar, Ashland, USA). For phenotyping of CMV specific T cells, PE labeled tetramer HLA/CMV epitope complex (HLA-A*01:01 CMV pp50: VTEHDTLLY; HLA-A*02:01 pp65-NLVPMVATV; HLAA*24:02 pp65-QYDPVAALF; HLA-B*07: 02 pp65-TPRVTGGGAM; HLA-B*08:01 IE1-ELRRKMMYM; HLA-B*35:01 pp65-IPSINVHHY, MBL International, Woburn, USA) was included in the panel with the above mentioned antibodies.