Human lymphocyte separation medium

Our study included four newly diagnosed CML patients who had undergone treatment with imatinib between 2017 and 2018 at the Department of Hematology, Beijing Hospital and the Department of Hematology, Beijing Tongren Hospital. One healthy donor was also included in our study. All patients were diagnosed according to the 2008 World Health Organization (WHO) consensus criteria. Peripheral blood mononuclear cells (PBMCs) for single cell RNA sequencing were isolated from 10 mL of peripheral blood within 2 h of the draw using Human Lymphocyte Separation Medium (DAKEWE; Shenzhen, China) according to the manufacturer's instructions. The clinical information is summarized in Supplementary Table 1.

Peripheral blood was obtained from the healthy donors. Blood sampling was performed following the required ethical procedures. Lymphocytes were isolated by density gradient centrifugation following the manual of the Human Lymphocyte Separation Medium (DAKEWE). Human T cells were purified using the human CD3 T cell isolation kit (BioLegend), stimulated with CD3/CD28 T cell Activator Dynabeads (Gibco) and cultured at 10⁶/mL in X-VIVO 15 Serum-free Hematopoietic Cell Medium (Lonza), supplemented with 5 ng/mL human IL-7 (PeproTech) and 5 ng/mL human IL-15 (PeproTech)^{40 (link),65 (link)}, with slight modification. 48 h after T cell stimulation, T cells were transduced with lentiviral supernatants from 293 T cell line in the presence of polybrene (6 μg/mL, Yeasen) by centrifugal infection. T cells were analyzed by flow cytometry 4 days after transduction and were used for further experiments. All human subjects were informed and signed informed consent prior to inclusion in the study and all human cell isolation and related experiments were approved by the Ethics Committee for Human Studies of Second Affiliated Hospital of Zhejiang University School of Medicine (2019 NO.388).

The Peripheral Blood Mononuclear Cells (PBMCs) were isolated from 20 ml of blood samples using human lymphocyte separation medium (Dakewe, Beijing, China) according to the the manufacturer’s recommendations. For T cell proliferation assay and cytokines production assay, the isolated PBMCs were freshly cultured in RPMI-1640 containing 20% fetal bovine serum. For elispot assay, the PBMCs were frozen in fetal bovine serum with 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until enough samples were collected.

Peripheral blood of healthy volunteers was drawn and placed onto human lymphocyte separation medium (Dakewe Biotech Co., Ltd). PBLs were isolated by differential density gradient centrifugation and plated in U-shaped bottom 96-well cell culture plates (2 × 10⁵ cells/well) using RMPI 1640 medium containing 10% FBS (Invitrogen, Carlsbad, CA, USA). Antibodies against CD3 (16–0037-85, eBioscience) and CD28 (16–0289-85, eBioscience) were added into each well (2 μg/mL). After 72 h, PBLs were dyed with fluorescence-conjugated antibodies against CD3 (300,308, BioLegend), CD8 (300,906, BioLegend) and CD25 (302,610, BioLegend) and sorted by flow cytometer (MoFlo XDP, Beckman Coulter, Inc.). Activated T cells (CD3⁺CD8⁺CD25⁺) were collected.

Peripheral blood of healthy donors was drawn through routine venipuncture. Peripheral blood lymphocytes (PBLs) were isolated using human lymphocyte separation medium (Dakewe Biotech Co, Ltd) through differential density gradient centrifugation. Cells were plated in U-shaped bottom 96-well cell culture plates (2 × 10⁵ cells/well) using the Roswell Park Memorial Institute 1640 medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) at 37℃. Antibodies against CD3 (16-0037-85, eBioscience) and CD28 (16-0289-85, eBioscience) were added into the media at a concentration of 2 μg/mL. After 72 hours, PBLs were dyed with fluorescently conjugated antibodies against CD3 (300 308, BioLegend), CD8 (300 906, BioLegend), and CD25 (302 610, BioLegend) and then sorted by flow cytometer (MoFlo XDP, Beckman Coulter, Inc). Activated T cells (CD3⁺CD8⁺CD25⁺) were collected.

Whole blood was taken from patients and collected in Vacutainer tubes containing EDTA. Blood samples were processed within 12 h of collection. RBC was separated by using the density gradient method. First, the blood was separated by centrifugation (Eppendorf, Centrifuge 5810R) at 150 g for 10 min, and the supernatant was removed. Then, an equal volume of PBS was added to the precipitate and mixed gently. The diluted precipitate was then slowly added dropwise to 1.5 times the precipitate volume of the Human Lymphocyte Separation Medium (Dakewe Biotech, China) and centrifuged at 800 g for 15 min then slowly removed the supernatant. Then, an equal volume of PBS was added to the precipitate, mixed gently, and then centrifuged at 150 g for 15 min. 3× volume of RBC lysis buffer was added to purified RBCs, and the supernatant lysate was collected after centrifugation. DNA from RBC was extracted using the FastPure Blood/Cell/Tissue/Bacterial DNA Isolation Mini Kit (Vazyme, China) based on the principle of affinity column. The RBC DNA of patient LC10 with lung cancer was also extracted with Nextractor Whole Blood DNA Kit (Genolution, Korea) based on the principle of magnetic beads, according to the manufacturer's protocol.