Peripheral venous blood samples were collected in heparin-containing tubes, and PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque Plus solution (Amersham Biosciences, Uppsala, Sweden). pDCs and cDCs were identified phenotypically as Lin1-HLA-DR+CD123+ cells and Lin1-HLA-DR++CD11c+ cells by flow cytometry, as previously described (21 (link)).
Ficoll paque plus solution
Manufactured by Cytiva
Sourced in Sweden, United Kingdom, United States
Ficoll-Paque Plus solution is a density gradient medium used for the separation and isolation of cells and cellular components. It is composed of a mixture of Ficoll polymer and sodium diatrizoate, which creates a density gradient that allows for the efficient separation of different cell types during centrifugation.
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Lab products found in correlation
Facsaria flow cytometer, by BD (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Facs aria 1 sorter, by BD (2 mentions)
Anti hcd4 pe, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Hla dr percp, by BD (2 mentions)
Cd11c apc, by BD (2 mentions)
Rhtnf α, by Thermo Fisher Scientific (2 mentions)
Rhil 4, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cd56 microbeads, by Miltenyi Biotec (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
M csf, by R&D Systems (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Recombinant human il 4, by Thermo Fisher Scientific (1 mentions)
Mv2 basal medium, by PromoCell (1 mentions)
Growth supplement, by PromoCell (1 mentions)
Macs cell separation system, by Miltenyi Biotec (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Trypan blue solution, by Merck Group (1 mentions)
21 protocols using ficoll paque plus solution
1
Isolation and Identification of Dendritic Cells
2
Isolation and Sorting of Human NK Cells
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Peripheral venous blood samples were collected in heparin-containing tubes, and PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque Plus solution (Amersham Biosciences, Uppsala, Sweden). NK cells were phenotypically identified as CD3-CD56+ cells by flow cytometry, as previously described [21 (link),22 (link)]. NK cells were isolated using CD56 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD3-CD56+ cells was greater than 95% as analyzed by flow cytometry. For sorting of CD69+ and CD69- NK cells, PBMCs were stained with APC-conjugated anti-CD3, FITC-conjugated anti-CD56, PerCP-conjugated anti-CD45, and PE-conjugated anti-CD69 mAb, and were sorted to obtain CD45+CD3-CD56+CD69+ and CD45+CD3-CD56+CD69- NK cells using a FACS Aria I sorter (BD Biosciences, Mountain View, CA) at purities of > 98%.
3
Identification of MAIT and NKT Cells
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Peripheral venous blood samples were collected in heparin-containing tubes, and PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque Plus solution (Amersham Biosciences, Uppsala, Sweden). MAIT and NKT cells were identified phenotypically as CD3+TCRγδ-Vα7.2+CD161high and CD3+6B11+ cells, respectively, by flow cytometry, as previously described (17 (link), 18 (link), 19 (link), 20 (link), 21 (link)).
4
Identification of MAIT, NK, and NKT Cells
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Peripheral venous blood samples were collected into heparin-containing tubes, and PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque Plus solution (Amersham Biosciences, Uppsala, Sweden). MAIT, NK, and NKT cells were identified phenotypically as CD3+TCRγδ−Vα7.2+CD161high cells, CD3−CD56+ cells, and CD3+TCRVα24-Jα18+ cells, respectively, by flow cytometry as previously described (27 (link)28 (link)29 (link)).
5
Isolation and Stimulation of PBMCs
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Peripheral blood samples were collected into ethylenediaminetetraacetic acid-treated tubes by venipuncture from the subjects after an 8-h fasting and were layered on the Ficoll-Paque Plus solution (Amersham Biosciences, Amersham, Bucks, UK) in a centrifuge tube, centrifuged at 400 × g for 20 min at 21°C, and peripheral blood mononuclear cells (PBMCs) were harvested. Then, divalent cation-free Hanks balanced salt solution was used for washing of cells at 300 × g for 5 min at 4°C. PBMCs were resuspended at 106 cells/ml in RPMI-1640 medium and prepared for the following procedures.
Freshly processed human PBMCs were stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate and 500 ng/ml of ionomycin in the presence of 5 μg/ml brefeldin A for 5 h at 37°C as described by others.[29 (link)] The cells were harvested and stained with anti-hCD4-PE (BD Biosciences, San Jose, California, USA) and anti-hCD8-Percp (BD Biosciences) for 30 min at room temperature, followed by staining with anti-hIL-17A-FITC (eBioscience, San Diego, California, USA) and anti-hIFN-γ-APC (eBioscience) after fixation and permeabilization. CD8+ subpopulations were determined using FACS-Calibur (BD Biosciences). A total of 1 × 105 events were collected for each subject and data were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).
Freshly processed human PBMCs were stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate and 500 ng/ml of ionomycin in the presence of 5 μg/ml brefeldin A for 5 h at 37°C as described by others.[29 (link)] The cells were harvested and stained with anti-hCD4-PE (BD Biosciences, San Jose, California, USA) and anti-hCD8-Percp (BD Biosciences) for 30 min at room temperature, followed by staining with anti-hIL-17A-FITC (eBioscience, San Diego, California, USA) and anti-hIFN-γ-APC (eBioscience) after fixation and permeabilization. CD8+ subpopulations were determined using FACS-Calibur (BD Biosciences). A total of 1 × 105 events were collected for each subject and data were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).
6
Isolation and Identification of MAIT and NKT Cells
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Peripheral venous blood samples were collected in heparin-containing tubes, and PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque Plus solution (Amersham Biosciences, Uppsala, Sweden). MAIT and NKT cells were identified phenotypically as CD3+TCRγδ−Vα7.2+CD161high and CD3+6B11+ cells, respectively, by flow cytometry, as previously described (14 (link)).
7
Osteoclast Differentiation from PBMCs
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Peripheral mononuclear blood
cells (PBMCs) were used as precursors of osteoclasts. The PBMCs were
obtained from fresh human blood donated by healthy male according
to the donation protocol approved by the Human Subjects Committee
of the University of Turku and Tokyo Medical and Dental University
as previously described.3 (link),26 (link) Blood was collected
into the tubes with heparin as an anticoagulation factor. The anticoagulated
blood was diluted 1:1 (v/v) with phosphate buffered saline (PBS),
layered over the Ficoll–Paque Plus solution (Amersham Pharmacia
Biotech, Uppsala, Sweden), and centrifuged at 1500 rpm for 15 min.
The buffy coats were collected and washed twice with PBS and resuspended
in a cell culture medium (α-MEM) containing 10% FBS, 1000 U/mL
penicillin–streptomycin. The cells were placed onto the specimens
at a density of 1 × 106 cells/cm2 and cultured
in the cell culture medium with an addition of 20 ng/mL RANKL (Peprotech
310-01) and 10 ng/mL M-CSF (R&D, 216-MC) for 14 days. Half of
the media in each sample were changed every 3 to 4 days.
cells (PBMCs) were used as precursors of osteoclasts. The PBMCs were
obtained from fresh human blood donated by healthy male according
to the donation protocol approved by the Human Subjects Committee
of the University of Turku and Tokyo Medical and Dental University
as previously described.3 (link),26 (link) Blood was collected
into the tubes with heparin as an anticoagulation factor. The anticoagulated
blood was diluted 1:1 (v/v) with phosphate buffered saline (PBS),
layered over the Ficoll–Paque Plus solution (Amersham Pharmacia
Biotech, Uppsala, Sweden), and centrifuged at 1500 rpm for 15 min.
The buffy coats were collected and washed twice with PBS and resuspended
in a cell culture medium (α-MEM) containing 10% FBS, 1000 U/mL
penicillin–streptomycin. The cells were placed onto the specimens
at a density of 1 × 106 cells/cm2 and cultured
in the cell culture medium with an addition of 20 ng/mL RANKL (Peprotech
310-01) and 10 ng/mL M-CSF (R&D, 216-MC) for 14 days. Half of
the media in each sample were changed every 3 to 4 days.
8
Flow Cytometric Analysis of Peripheral Blood Mononuclear Cells
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Peripheral blood samples were drawn in ethylenediaminete-traacetic acid tubes from all participants and separated to peripheral blood mononuclear cells (PBMC) by centrifugation on Ficoll-Paque Plus solution (Amersham Biosciences, Amersham, Bucks, UK), at 400×g for 20 min at 21 °C. Then, PBMCs were washed by divalent cation-free Hanks balanced salt solution at 300×g for 5 min at 4 °C and resuspended at 106 cells/ml in RPMI-1640 medium.
For cytokine analysis, freshly processed human PBMCs were stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate and 500 ng/ml of ionomycin and incubated at 37 °C in the presence of 5 μg/ml Brefeldin A. After 5 h cells were collected and stained as previously demonstrated with anti-hCD4-PE (BD Biosciences, San jose, California, USA) for 30 min at room temperature. For detection of intracellular cytokines, cells were subsequently stained with anti-hIL-17-FITC (eBioscience, San Diego, California), anti-hIFN-γ-FITC (eBioscience) after fixation and permeabilization. Cells were analyzed by FACS-Calibur (BD Biosciences) and isotype control was used to set gates. A total of 1 × 106 events were examined for each subject. The data were presented using proportions of cells and were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).
For cytokine analysis, freshly processed human PBMCs were stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate and 500 ng/ml of ionomycin and incubated at 37 °C in the presence of 5 μg/ml Brefeldin A. After 5 h cells were collected and stained as previously demonstrated with anti-hCD4-PE (BD Biosciences, San jose, California, USA) for 30 min at room temperature. For detection of intracellular cytokines, cells were subsequently stained with anti-hIL-17-FITC (eBioscience, San Diego, California), anti-hIFN-γ-FITC (eBioscience) after fixation and permeabilization. Cells were analyzed by FACS-Calibur (BD Biosciences) and isotype control was used to set gates. A total of 1 × 106 events were examined for each subject. The data were presented using proportions of cells and were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).
9
Isolation and Characterization of Myeloid Dendritic Cells
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Bone marrow samples from patients with SAA and HCs were collected in heparin anticoagulant tubes. Bone marrow mononuclear cells were isolated by density gradient centrifugation using Ficoll‐Paque Plus solution (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions. Thereafter, we placed the cells in a culture medium containing 79% RPMI 1640 (Gibco‐BRL, Grand Island, NY, USA), 20% foetal bovine serum (Gibco‐BRL), and 1% penicillin–streptomycin (Hyclone, South Logan, UT, USA) at a cell concentration of 1.5 × 106/mL. After incubation for 2 h, the suspended cells were discarded, and the remaining semi‐adherent cells were cultured in a medium containing 100 ng/mL recombinant human granulocyte monocyte colony stimulating factor (rhGM‐CSF) (North China Pharmaceutical Co., Hebei, China) and 25 ng/mL recombinant human (rhIL‐4) (PeproTech, Rocky Hill, NJ, USA) at 37°C with 5% CO2. The medium was changed, and the above‐mentioned cytokines were added every 2 days. On Day 6, 1000 U/mL rhTNF‐α (PeproTech) was added to induce mDC maturation. On Day 7, the suspended cells were collected, labelled with APC‐CD11c (BD Pharmingen) and PerCP‐HLA‐DR (BD Pharmingen), and sorted using a FACSAria flow cytometer (BD Biosciences, San Jose, CA, USA). Purity of the mDCs was measured and quantified as the percentage of CD11c+HLA‐DR+ cells among all sorted cells.
10
Isolation and Purification of CD4+ and CD8+ T Cells
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Peripheral blood samples were collected in heparin anticoagulant tubes, and mononuclear cells were isolated by density gradient centrifugation using Ficoll‐Paque Plus solution (Amersham Biosciences). An anti‐CD4 or anti‐CD8 monoclonal magnetic bead antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) was added to the mononuclear cell suspension and incubated at 4℃ in dark for 20 min. CD4‐ or CD8‐positive lymphocytes were selected using an MS type sorting column according to the manufacturer's instructions (Miltenyi Biotec GmbH). The purity of the separated cells was determined using flow cytometry.
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