Ficoll density gradient centrifugation

The CML cell lines K562 and K562/G01 (Cell Repository, Chinese Academy of Science, Shanghai, China) was incubated at 37°C, with 95% air and 5% CO₂ in RPMI 1640 (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco). The human CML cell line KU812 (Basic Medical Institute, Chinese Academy of Medical Sciences, Beijing, China) was incubated at 37°C, with 95% air and 5% CO₂ in RPMI 1640 containing 10% FBS. The human Ph+ ALL cell line SUP-B15 (Cell Repository, Chinese Academy of Science, Shanghai, China)was incubated at 37°C, with 95% air and 5% CO₂ in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) containing 10% FBS. The human renal epithelial cell line 293T (Cell Repository, Chinese Academy of Science, Shanghai, China) was cultured at 37°C, with 95% air and 5% CO₂ in DMEM (Gibco) containing 10% FBS. Ficoll density gradient centrifugation (TBD Science, Tianjin, China) isolate blast cells. The K562/G01 cells were transfected with Bcl-6 siRNA using Lipofectamine 2000 (Invitrogen, USA) for 48 h. Bcl-6 siRNA was synthesized by GenePharmagps in Shanghai. The sequences for the Bcl-6 siRNA1 were:5′-GCAGUUUAGAGCCCAUAAATT-3′ (sense), 5′-UUUAUGGGCUCUAAACUGCTT-3′ (antisense). Those for Bcl-6 siRNA2 were: 5′-GCAAUAUCUAUUCACCCAATT-3′ (sense), 5′-UUGGGUGAAUAGAUAUUGCTT-3′ (antisense).

The human Ph+ ALL cell line SUP-B15 (the American Type Culture Collection, CRL-1929), CML cell line K562 (Hematology Lab, West China Hospital of Sichuan University, Sichuan, China 610041), and primary leukemic blasts were maintained in RPMI 1640 or IMDM (Hyclo, USA), supplemented with 5% penicillin/streptomycin and 10% fetal bovine serum (FBS) in a humidified atmosphere at 37°C in a 5% CO2 incubator. Blast cells were isolated by Ficoll density gradient centrifugation (TBD Science, Tianjin, China). Ribavirin (Sigma, USA) was dissolved in H₂O at a stock concentration of 20mmol/L and sterile filtered, kept at -80°C in small aliquots. Imatinib (Basel, Switzerland), U0126 (Beyotime), and CGP57380 (Sigma) were prepared in dimethyl sulfoxide (DMSO) stored at -20°C with 10 mmol/L and diluted into suitable concentrations before being used.

Uteri and placentae were carefully separated from pregnant female mice after euthanasia on Gd 14 and were washed twice in ice-cold PBS. Tissues were then cut into 1–3 mm pieces, and single-cell suspensions were obtained by filtration through 48 µm sterile nets. Mononuclear cells were obtained using Ficoll density gradient centrifugation in mouse lymphocyte separation medium (TBD Science, China) and used for flow cytometry analysis.

Lung and spleen tissues were collected from mice of all five groups. After washing with RPMI medium was repeated for 3–4 times, lung tissue was fragmented and digested with 1 mg/mL collagenase type IV (Cat# 8160; Sigma-Aldrich, USA) and 0.2 mg/mL DNase (Cat# AMDP1; Sigma-Aldrich, St. Louis, USA) at 37°C for 30 min. Digested lung tissue was then filtered through a sterile 48-micron mesh filter and washed twice with cold PBS. Subsequently, mononuclear cells were obtained by Ficoll density gradient centrifugation (Cat# LTS1092; TBD Science, China) and further used in flow cytometry analysis.
Spleen tissues were immediately filtered through a 70-μm cell strainer, washed twice with PBS, and then lysed with a Red Blood Cell Lysis Buffer (Cat# R1010; Solarbio, China). Single cells from spleen tissue were collected and further used in flow cytometry analysis.

The paraffin-embedded tissue samples from 35 cases of T-cell lymphomas and 15 reactive hyperplasia of lymph node (RHL) were collected from Shandong Provincial Hospital affiliated to Shandong University prior to therapeutic intervention in this study. All patients were diagnosed according to the WHO criteria between January 2009 and July 2013. Of the 35 cases of T-cell lymphomas (23 males and 12 females; age range 25–82 years, median55 years), 12 were peripheral T-cell lymphoma non-specific (PTCL-NOS), 6 were angioimmunoblasic T-cell lymphoma (AITL), 6 were CTCL, 4 were enteropathy associated T-cell lymphoma (EATL), 7 were NK/TCL. Peripheral blood of healthy volunteers was drawn by heparin anticoagulation, and peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation (TBD Science, Tianjin, China). From this fraction, T cells were isolated by Nylon Wool Fiber Columns. This study was approved by the Medical Ethical Committee of Shandong Provincial Hospital affiliated to Shandong University. All human samples were obtained after informed consents had been given, according to the Declaration of Helsinki.

Mice uteri and placentas were carefully dissected from pregnant mice at gd 11 and were washed twice in cold PBS. Tissues were then cut into small pieces and prepared for cell dispersion by GentleMACS dissociator (Miltenyi Biotech). Sterile nets (48 µm) were used to obtain single cell suspensions. Mononuclear cells were collected from the white film layer after Ficoll density gradient centrifugation in mouse lymphocyte separation medium (TBD Science), and were then analyzed with flow cytometry. The mice cadavers were stored in a -20°C freezer and disposed of by professional organizations.