Approximately 30ml of KTR blood was collected by venesection in Lithium-Heparin vacuum tubes and processed within 2h. Plasma was collected from centrifuged blood (2000g, 10mins, no brake) and stored at −80°C. The KTR PBMC’s were separated by Lymphoprep™ (Oslo, Norway) centrifugation techniques, and re-suspended in either Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, New York, USA) supplemented with 70% (v/v) foetal calf serum (FCS, Bovogen, Kielor East, Australia), 1mM L-glutamine (Sigma, Sao Paulo, Brazil), 1mM Penicillin (Commonwealth Serum Laboratories (CSL), Parkville, Australia)/ 1mM Gentamycin (Gibco®, Grand Island, New York) and 10% (v/v) Dimethyl sulfoxide (DMSO, VWR International, Fontenay-sous-Bois, France) for cryopreservation media or resuspended in RPMI 1640 supplemented with 20% (v/v) FCS, 1mM L-glut., 1mM Pen/Gent and placed in 5% CO2, 37°C incubator, overnight. Viable cell concentrations were determined via Trypan-blue (BDH Chemicals, Poole, England) exclusion assay.
Lymphoprep
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Sweden
Lymphoprep is a density gradient medium used for the isolation of mononuclear cells, such as lymphocytes and monocytes, from whole blood or bone marrow samples. It works by separating the different blood cell components based on their density during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Lymphoprep, by STEMCELL (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Merck Group (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by Avantor (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Vacutainer, by BD (1 mentions)
Complete edta free, by Roche (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by GoldBio (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Hepes ph 7, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
15 protocols using lymphoprep
1
Cryopreservation and Culture of KTR PBMCs
2
Isolation of Bovine PBMC for RNA Analysis
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Blood samples were collected from the jugular vein of the dairy cows into 10 mL K2-EDTA vacutainer tubes (BD Vacutainer, Becton Dickinson Co., Franklin Lakes, NJ, USA). Two tubes were collected from each animal to obtain 15 to 20 mL of blood, and the samples were immediately placed on ice and transported to the laboratory for the isolation of PBMC within 30 min of the time of sampling.
The PBMCs were isolated using density gradient centrifugation. Briefly, whole blood samples were diluted with phosphate-buffered saline (PBS) in a 1:1 ratio in 15 mL conical tubes. Then, 4 mL of Lymphoprep (STEMCELL Technologies lnc., Vancouver, BC, Canada) was added to the new tubes, and 8 mL of the diluted blood samples were overlaid on the Lymphoprep. After centrifugation for 20 min at 800× g and at 22 °C, the layer of cells above the Lymphoprep was collected and washed twice with PBS to obtain purified PBMCs that were then suspended in 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and transferred to a 1.5 mL tube. The PBMCs were immediately stored at −80 °C until the RNA isolation process.
The PBMCs were isolated using density gradient centrifugation. Briefly, whole blood samples were diluted with phosphate-buffered saline (PBS) in a 1:1 ratio in 15 mL conical tubes. Then, 4 mL of Lymphoprep (STEMCELL Technologies lnc., Vancouver, BC, Canada) was added to the new tubes, and 8 mL of the diluted blood samples were overlaid on the Lymphoprep. After centrifugation for 20 min at 800× g and at 22 °C, the layer of cells above the Lymphoprep was collected and washed twice with PBS to obtain purified PBMCs that were then suspended in 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and transferred to a 1.5 mL tube. The PBMCs were immediately stored at −80 °C until the RNA isolation process.
3
Lymphocyte Isolation from Whole Blood
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Whole blood was diluted with saline 1:1 and lymphocytes were isolated using lymphoprep (Invitrogen, UK) according to the manufacturer's protocol. The isolated lymphocytes were re-suspended in RPMI medium for further use.
4
Reagent Procurement for Protein Purification
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Isopropyl β-D-1-thyogalacto-pyranoside and kanamycin were purchased from Gold Biotechnology, Inc., St Louis, MO, USA. Complete EDTA-free was from Roche (Mannheim, Germany) and Bradford reagent was from Bio-Rad (Hercules, CA). Bovine serum albumin (BSA albumin fraction V) was provided by Sigma Aldrich (St. Louis, MO, USA). Lymphoprep and RPMI medium were purchased from Invitrogen, Thermo Scientific (Austin, TX, USA), while L-glutamine was form Gibco Thermo Scientific (Austin, TX, USA). All other agents used were of analytical grade.
5
Isolation and Preservation of NK Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated using a Lymphoprep™ (Gibco) centrifugation gradient and half of the PBMCs were used directly for NK cell enrichment as previously described [21 (link),22 (link)]. NK cells were kept at a concentration of 106 NK cells/ml in media (RPMI-1640 with L-Glutamine (Gibco) with 10% heat-inactivated fetal bovine serum (FBS; Gibco) and 1% Penicillin/Streptomycin (Gibco)) until analysis. The other half of the PBMCs was frozen based on a method by Paich et. al. [23 (link)]).
6
Isolation and Culture of Mononuclear Cells
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Peripheral venous blood was collected from subjects into syringes containing 5 U/ml heparin. Mononuclear cells were isolated by differential centrifugation (800 g, 30 min, 20°C) over Lymphoprep and washed twice with sterile phosphate-buffered saline (PBS; GIBCO, Paisley, UK) at 500 g (5 min, 20°C). Cells were resuspended in 10 ml RPMI-1640 medium (Invitrogen) supplemented with 100 U/ml of penicillin (GIBCO), 100 µg/ml streptomycin (GIBCO) and 20 mM HEPES pH 7.4 (Sigma-Aldrich), and plated at a density of approximately 5×106 cells/ml in 8 cm2 Nunclon™ Surface tissue culture dishes (Nunc, Roskilde, Denmark) at 37°C, 5% CO2. After 2 h, non-adherent cells were discarded and 10 ml of fresh RPMI supplemented with 10% foetal bovine serum (FBS; Sigma) added to each tissue culture dish. Cells were then cultured for 5 days at 37°C, 5% CO2, with the addition of a further 10 ml of fresh 10% FBS/RPMI after 24 h. Cells were then washed twice in PBS, scraped and spun down at 500 g (5 min, 20°C). Cells were resuspended into X-vivo-15 medium (Cambrex, MD, USA) and plated at a density of 106 cells per 8 cm2 Nunclon™ dish for a further 25 h at 37°C, 5% CO2.
7
Allogeneic Stem Cell Transplant Monitoring
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All experiments were performed with authorization of the Institutional Review Board (IRB) for Human Research at the Catholic University of Korea. Study patients were the recipients of allogeneic SCT that were initially diagnosed with one of the hematologic diseases designated by the World Health Organization (WHO). Heparinized blood samples were collected from the recipients at a time of high CMV DNAemia for those diagnosed with CMV viremia (CMV+ group) and at any time after transplantation for those without CMV viremia (CMV- group). In addition, blood samples were collected from the recipients before conditioning (pre-HSCT group) and healthy donors (healthy donor group) before harvesting hematopoietic stem cells.
Mononuclear cells were isolated by overlaying the heparinized blood samples on a Ficoll-Hypaque gradient (density, 1.077; Lymphoprep; Gibco-BRL, Carlsbad, CA, USA), followed by centrifugation at 400×g for 30 minutes. The buffy coats were harvested and washed twice with phosphate-buffered saline (pH 7.4).
Mononuclear cells were isolated by overlaying the heparinized blood samples on a Ficoll-Hypaque gradient (density, 1.077; Lymphoprep; Gibco-BRL, Carlsbad, CA, USA), followed by centrifugation at 400×g for 30 minutes. The buffy coats were harvested and washed twice with phosphate-buffered saline (pH 7.4).
8
Isolation and Purification of Neutrophils from Human Blood
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Three mL Lymphoprep (STEMCELL Technologies, Saint Égrève, France) were layered onto 3 mL Histopaque 1119 (Sigma-Aldrich) and 8 mL blood anticoagulated with EDTA were carefully layered on top. After centrifugation at 700 × g for 30 min with minimum speed for acceleration and break, the polymorphonuclear cells were recovered at the interface between Histopaque-1119 and Lymphoprep, transferred to a new tube and washed with 1X PBS (Gibco, Life Technologies). Contaminant erythrocytes were lysed with 1 mL Cell Lysis Solution (Promega Biotech Iberica S.L.) for 10 min before centrifuging at 160 × g for 10 min. All procedures were conducted at room temperature (RT). Neutrophil viability was determined by trypan blue (Sigma-Aldrich) exclusion. Neutrophil purity and count were determined by extension and staining, by flow cytometry and by quantification with the automatic hematology analyzer Sysmex® Serie-XN- BF.
9
Isolation and Expansion of Human γδ T Cells
10
Generation of Monocyte-Derived Dendritic Cells
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Immature moDCs were generated from monocytes obtained from human peripheral blood mononuclear cells (PBMCs) isolated from buffy coats of healthy donors (Sanquin) by a sequential Lymphoprep (Thermo Fisher Scientific, Waltham, MA, USA) and Percoll (GE Healthcare, Chicago, IL, USA) gradient and cultured for 5–6 days in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Lonza, Basel, Switzerland), 100 U/mL penicillin/streptomycin (Lonza, Basel, Switzerland), and 2 mM glutamine (Lonza, Basel, Switzerland) (complete RPMI 1640) in the presence of recombinant human IL-4 (500 U/mL) and GM-CSF (800 U/mL) (ImmunoTools, Friesoythe, Germany). The THP-1 and JY cell lines were cultured in complete RPMI 1640, and the Mel-Juso and Mel-BRO cell lines were cultured in complete Iscove’s modified Dulbecco’s medium (IMDM). The gp100-specific HLA-DRB∗0401-restricted T cell line Bridge gp:44 B855 (link) and the retroviral T cell receptor (TCR)αβ-transduced T cell clone specific for the gp100280–288 HLA-A2 minimal epitope56 (link) were cultured in Yssel’s medium57 (link) supplemented with 1% human serum, penicillin, streptomycin, and glutamine.
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