Hs5 human bone marrow stromal cell line

The HS5 human bone marrow stromal cell line immortalized by E6/E7 (11 (link)), was obtained from American Type Culture Collection (Manassas, VA, USA). A total of 62 patients (male and female; aged 38–85 years) diagnosed with typical B-CLL were recruited from the First Affiliated Hospital of Nanchang University (Nanchang, China) in the present study. The diagnosis was based on clinical criteria and laboratory features, according to National Cancer Institute Criteria (19 (link)). All patients provided written informed consent, and the present study was approved by the ethics committee of The First Affiliated Hospital of Nanchang University. In all experiments, CLL cells were isolated from peripheral blood samples, which were collected from the patients, by density gradient centrifugation (20 (link)). Briefly, 5 ml blood was slowly added to a Falcon tube containing 6 ml pre-warmed Fico/Lite LymphoH buffer (Atlanta Biological, Lawrenceville, GA, USA) and then centrifuged at 435 × g for 20 min at room temperature. The isolated CLL cells was washed with pre-warmed PBS and incubated in RPMI-1640 medium (Cellgro; Mediatech, Inc., Hendon, VA, USA) supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 µg/ml; all from Cellgro; Mediatech, Inc.) overnight prior to experimentation.