Apc anti human cd4 clone rpa t4

ATL patient PBMCs were thawed and washed with PBS containing 0.1% BSA. To discriminate dead cells, we used the LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). For cell surface staining, cells were stained with APC anti-human CD4 (clone: RPA-T4) (BioLegend) and anti-SynCAM (TSLC1/CADM1) mAb-FITC (MBL) antibodies for 30 minutes at 4°C followed by a wash with PBS. HTLV-1 infected cells (CADM1+ and CD4+) were sort-purified with FACS Canto (Beckman Coulter) to reach 98–99% purity. Data was analyzed by FlowJo software (Treestar). Soon after the sorting, 10000-50000 HTLV-1 infected cells were centrifuged and used for ATAC-seq as previously described [5 (link)]. Total RNA was isolated from the remaining cells using the RNeasy Mini Kit (Qiagen). Library preparation and high-throughput sequencing were performed by Macrogen Inc. (Seoul, Korea). The diagnostic criteria and classification of clinical subtypes of ATL were performed as previously described [28 ]. 77 ATAC-seq datasets from 13 human primary blood cell types and datasets from 42 AML patients were obtained from the Gene Expression Omnibus (GEO) with accession number GSE74912 [7 (link)]. ATAC-seq datasets from 7 CLL patients were obtained from GSE111015 [18 (link)] and RNA-seq datasets of CD4⁺T and Mono cells were obtained from GSE74246 [7 (link)].

APC antihuman CD4, clone RPA-T4 was purchased from BioLegend. FITC antihuman CD3, clone SK7; PE antihuman CD8, clone SK1; PE antihuman CD279, clone MIH4; APC antihuman CD273, clone MIH18; and PE antihuman CD274, clone MIH1 were purchased from BD (Franklin Lakes, NJ, USA) To determine cell viability, cells were stained with 7-amino-actinomycin D (7-AAD, BD Pharmingen). Flow cytometry was performed using FACSCantoII or FACSLyric (both from BD (Franklin Lakes, NJ, USA). Data were analyzed using FACSDiva software (BD Biosciences).