A total of 7.5 mL of PB from each HD or BC was collected in the morning of their surgery using Vacutainer ACD tubes (NIPRO Corporation, Japan, Osaka) containing heparin. The collected PB was immediately placed in 10 mL Ficoll-Hypaque (SIGMA-ALDRICH, London, UK), and mononuclear cells were isolated by density centrifugation (500×g, 30 min, 20 °C). The cells were washed with phosphate-buffered saline (PBS) for 5 min at 300×g at 4 °C. Around 2.5 to 5 × 106 PBMCs were transplanted intravenously into 8‒12-week-old NOG or NOG-hIL-4-Tg mice. The number of mice transplanted depended on the amount of available material, in particular the BC PBMCs. The mice transplanted with HD PBMCs were referred to as HD-M, and those transplanted with BC PBMCs as BC-M.
Vacutainer acd tubes
Manufactured by Nipro
Sourced in Japan
Vacutainer ACD tubes are blood collection tubes used for the separation and preservation of whole blood samples. They contain an anticoagulant solution of sodium citrate, citric acid, and dextrose, which prevents the blood from clotting during collection and storage.
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Lab products found in correlation
7 protocols using vacutainer acd tubes
1
Xenotransplantation of Human PBMCs
2
Mononuclear Cell Isolation from Human Blood
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RPMI 1640 medium and supplements were purchased from Nissui, Co., Ltd., (Tokyo, Japan) and peripheral blood (PB; 50 mL) was collected from each HD in the morning using Vacutainer ACD tubes (NIPRO Corporation, Osaka, Japan) containing heparin. The collected PB was immediately transferred to a 10-mL Ficoll–Hypaque density gradient medium (Sigma-Aldrich, London, UK) and centrifuged (500× g, 30 min, 20 °C) to isolate mononuclear cells. The remaining erythrocytes were removed by osmotic lysis. The cells were washed with phosphate-buffered saline (PBS) for 5 min at 300× g, 4 °C, and the cell number was counted.
3
Isolation of Mononuclear Cells from Peripheral Blood
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RPMI 1640 medium and supplements were purchased from NISSUI; 50 mL of peripheral blood (PB) was collected from each healthy donor in the morning using Vacutainer ACD tubes (NIPRO Corporation) containing heparin. The collected blood was immediately transferred to 10 mL of density gradient medium Ficoll‐Hypaque (Sigma‐Aldrich), and centrifuged (500 × g, 30 min, 20°C) to isolate mononuclear cells. The remaining erythrocytes were removed through osmotic lysis. The cells were washed with phosphate‐buffered saline (PBS) for 5 min at 300 × g, 4°C, and the cell number was estimated.
4
Engrafting Human PBMCs in NOG Mice
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A total of 7.5 mL of PB from each HD or BC was collected in the morning of their surgery using Vacutainer ACD tubes (NIPRO Corporation, Japan, Osaka) containing heparin. The collected PB was immediately placed in 10 mL Ficoll-Hypaque (SIGMA-ALDRICH, London, UK), and mononuclear cells were isolated by density centrifugation (500 × g, 30 min, 20 °C). The cells were washed with phosphate-buffered saline (PBS) for 5 min at 300 × g at 4 °C. Around 2.5 to 5 × 10 6 PBMCs were transplanted intravenously into 8-12-week-old NOG or NOG-hIL-4-Tg mice. The number of mice transplanted depended on the amount of available material, in particular the BC PBMCs. The mice transplanted with HD PBMCs were referred to as HD-M, and those transplanted with BC PBMCs as BC-M.
5
Isolation of PBMCs from Blood Samples
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Blood samples (7.5 ml) were collected both from healthy donors and from patients on the morning of their surgery using Vacutainer ACD tubes (NIPRO Corporation, Japan, Osaka). PBMCs were then isolated from the collected blood by density centrifugation at 5000g for 30 min at 20 °C using Ficoll-Hypaque reagent (Sigma-Aldrich, London, UK) according to the manufacturer’s instructions. PBMCs were aspirated and washed with phosphate-buffered saline at 3000g for 5 min at 4 °C. All samples were processed within 12 h of collection.
6
Engrafting Human Immune Cells in Mice
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The healthy donors (HD) enrolled in the experiments and all of mice transplanted with HD PBMC are listed in Table 1. A total of 7.5 mL of PB from healthy donors was drawn into Vacutainer ACD tubes (NIPRO Corporation, Osaka, Japan) containing heparin. The collected PB was immediately placed in 10 mL of Ficoll-Hypaque (Sigma-Aldrich, London, UK), and mononuclear cells were isolated by density centrifugation (500 × g, 30 min, 20°C). The cells were washed with PBS for 5 min at 300 × g, 4°C. Numbers of PBMCs ranging from 2.5 to 5 × 10 6 were intravenously transplanted into 8-to 12-weekold NOG or NOG-hIL-4-Tg mice. Two mice from each strain were transplanted with the same donor PBMCs, to use one for peptide immunization and the other for negative control (PBS and Freund's adjuvant).
7
PBMC Isolation and Transplantation for GVHD Modeling
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A total of 7.5 ml PB from healthy donors was drawn into Vacutainer ACD tubes (NIPRO Corporation, Japan, Osaka) containing heparin. The collected PB was immediately placed in 10 ml of Ficoll-Hypaque (SIGMA-ALDRICH, UK, London), and mononuclear cells were isolated by density centrifugation (500 × g, 30 min, 20°C). The cells were washed with phosphate-buffered saline (PBS) for 5 min at 300 × g, 4°C. Doses of 2.5 to 5x106 PBMC were transplanted intravenously into 8- to 12-week-old NOG or NOG-hIL-4-Tg mice. For the GVHD assay, the mice were irradiated (2.5 Gy), and 1 day after irradiation, the PBMCs were transplanted. The body weights of the individual mice were measured weekly. The number of mice transplanted depended on the amount of available material, particularly the healthy donor (HD) PBMCs.
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