Blood samples were obtained from two EBV-seropositive healthy donors with informed consent under the approval of the Ethical Committee for Epidemiology of Hiroshima University. PBMCs were isolated from buffy coats with density centrifugation using lymphocyte separation medium 1077 (Takara Bio, Shiga, Japan). FACS-sorted CD4+ T cells or CD8+ T cells were plated at 1 × 105 cells per well in a U-bottom 96-well plate and co-cultured with autologous LCL irradiated at 30 Gy starting at 40:1 responder to stimulator ratio in RPMI-1640 supplemented with 10% FCS, 1% Penicillin-streptomycin, and 10 ng/mL human recombinant IL-7 (BioLegend, San Diego, CA, USA). An amount of 10 ng/mL IL-2 was added from day 2 of co-culture. Cells were then re-stimulated with irradiated autologous LCL at a ratio of 1:1 responder to stimulator weekly for a total of 5 stimulation rounds. Thereafter, T cells were expanded in T25 culture flasks on a weekly basis counting, and fed/split as necessary to give a final density of 1–2 × 106 T cells/cm2. All cell counts were given as viable cells via trypan blue exclusion of dead cells.
Lymphocyte separation medium 1077
Manufactured by Takara Bio
Sourced in Japan
Lymphocyte separation medium 1077 is a sterile, endotoxin-tested liquid medium designed for the isolation of lymphocytes from whole blood or other cell suspensions. It is based on a density gradient solution and allows for the separation of lymphocytes from other blood components through centrifugation.
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Lab products found in correlation
2 protocols using lymphocyte separation medium 1077
1
Expansion of EBV-specific T cells
2
PBMC Isolation and Preparation
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PBMC preparation was conducted on the same day as sampling. PBMCs were isolated from the whole-blood samples by density gradient centrifugation on Lymphocyte Separation Medium 1077 (TaKaRa
Bio, Shiga, Japan), according the manufacturer’s instructions. Whole-blood samples (10 ml) were diluted with an equal volume of phosphate-buffered saline (PBS), carefully layered onto the
Lymphocyte Separation Medium 1077 and centrifuged at 450 × g for 40 min at room temperature (23 ± 2°C). Thereafter, PBMCs were collected, and erythrocytes were lysed in
NH4Cl-base lysis buffer. The PBMCs were then washed twice with PBS and suspended at a concentration of 1 × 105 cells/ml in RPMI-1640 medium (Wako Pure Chemical
Industries, Osaka, Japan). The separated cells were used for proliferation assays and RNA extraction.
Bio, Shiga, Japan), according the manufacturer’s instructions. Whole-blood samples (10 ml) were diluted with an equal volume of phosphate-buffered saline (PBS), carefully layered onto the
Lymphocyte Separation Medium 1077 and centrifuged at 450 × g for 40 min at room temperature (23 ± 2°C). Thereafter, PBMCs were collected, and erythrocytes were lysed in
NH4Cl-base lysis buffer. The PBMCs were then washed twice with PBS and suspended at a concentration of 1 × 105 cells/ml in RPMI-1640 medium (Wako Pure Chemical
Industries, Osaka, Japan). The separated cells were used for proliferation assays and RNA extraction.
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