Ficoll hypaque density gradient centrifugation

The human GC cell line SGC7901 was purchased from the American Type Culture Collection (ATCC), and MKN45 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Amersham Pharmacia Biotech) of heparinized venous blood obtained from healthy volunteer donors at the Affiliated Hospital of Qingdao University (Qingdao, China), under the National Regulation of Clinical Sampling in China. Cells were maintained in RPMI 1640 culture medium (GIBCO/BRL) supplemented with 10% heat-inactivated FBS at 37°C in a humidified atmosphere with 5% CO₂.

10 ml peripheral venous blood was collected, of which a 3 ml sample was used for the detection of serum concentrations of cytokines, and the other 7 ml sample was used for CD4⁺ T cell isolation. According to the manufacturer's instructions, peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation (Amersham Biosciences, Piscataway, NJ, USA), and untouched CD4⁺ T cells were isolated from PBMCs by immunomagnetic beads (Miltenyi Biotec, Auburn, CA, USA). The purity of the CD4⁺ T cells was 94.1 ± 1.6% as evaluated by flow cytometry, and the cell vitality was 95.6 ± 1.5% as assessed by trypan blue staining. CD4⁺ T cells were harvested for flow cytometric analysis, RNA extraction, and γ-secretase inhibitor DAPT treatment.

The present study comprised 20 consecutive patients with primary MDS and 20 age-matched healthy controls. The characteristics of the patients are shown in Table 1. MDS patients were recruited from the Tianjin Cancer Hospital and Tangshan Hospital. Diagnoses were made according to the WHO criteria. All patients were clinically stable and untreated when the sample was collected. After a written informed consent was obtained, peripheral blood was collected in heparinized tubes from each patient and healthy control. Healthy controls (n = 20) were obtained from healthy volunteers in our hospital. For plasma collection, blood was centrifuged for 15 min at 1,000×g using a refrigerated centrifuge and stored at −80°C. PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation (Amersham Pharmacia Biotech, Piscataway, NJ, USA) following the protocol of the manufacturer. These cells were also freshly used for in vitro cytokine intervention experiment or were frozen in liquid nitrogen.

Human hepatoma cell lines HepG2 and Bel7402, normal hepatocyte line HL-7702, and human ovarian cancer cell line SKOV3 were purchased from American Type Culture Collection. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation (Amersham Pharmacia Biotech) from heparinized venous blood obtained from normal healthy volunteer donors at the Affiliated Hospital of Qingdao University (Qingdao, China). The cells were maintained in DMEM medium (GIBCO/BRL) supplemented with 10% heat-inactivated fetal bovine serum and cultured at 37°C in a humidfied atmosphere with 5% CO₂.

The HTLV outpatient clinic of the Institute of Infectious Diseases Emilio Ribas, São Paulo City, Brazil, has enrolled 727 individuals living with HTLV-1. This open cohort was created in 1997, and has been the place for some important studies and those regarding the characterization of IS [3 (link)]. This study was conducted during the period from January 2015 to January 2020.
All volunteers underwent serological screening for HTLV-1 at the Emilio Ribas Institute of Infectious Diseases (IIER), utilizing GOLD ELISA HTLV-1/2 (Diasorin, Dartford, UK), followed by confirmation with Western Blot (HTLV Blot 2.4^®, MP Diagnostics, Copenhagen, Denmark) and in-house nested PCR, a criteria to entry in the IIER HTLV Cohort [10 (link)]. Blood samples were collected in K3-EDTA (0.054 mL/tube) and plasma was obtained by centrifugation (15 min, 2500 rpm). Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-hypaque density gradient centrifugation (GE Healthcare Life, Austin, TX, USA). Cells were washed with saline solution, and cells were stored, as “dry pellet” at −80 °C. DNA was extracted from 1 × 10⁶ PBMCs using a commercial kit (Illustra Tissue and Cells Genomic Prep Mini Spin kit, Fairfield, CA, USA), according to the manufacturer’s instructions, and stored at −80 °C for HTLV-1 proviral load quantification.

AML bone marrow and peripheral blood samples were collected from study participants enrolled on the ETCTN/CTEP 10026 study (NCT02890329). Melanoma-infiltrating T cell were obtained from a participant in a phase I clinical trial at Dana-Farber Cancer Institute (DFCI) (NCT01970358)^{18 (link),42 (link)}. CAR T cells were obtained from a banked infusion product at DFCI^{35 (link)}. ALL bone marrow samples were collected at DFCI. All study participants provided written consent and samples were collected under Institutional Review Board-approved protocols at DFCI. Until the time of analysis, bone marrow and peripheral blood samples were stored in vapor-phase liquid nitrogen after Ficoll-Hypaque density gradient centrifugation (Cytiva, Cat. no. 17144002) and cryopreservation with 10% dimethyl sulfoxide (Sigma-Aldrich, Cat. no. D2650).