BMSCs were directly purchased from American Type Culture Collection (ATCC, #PCS-500-012TM, USA) and were cultured in the DMEM medium (Gibco, USA) following the ATCC’s instructions under the conditions with 5% CO2 atmosphere and 37 °C temperature. In addition, the EPCs were isolated and purified from human peripheral blood by using a Ficoll-Paque PLUS reagent (GE, USA) according to the previous work, which were subsequently cultured in vitro in the endothelial cell medium for 6 days. Then, cells were incubated with antibodies against CD34, CD133, and CD29, and the CD34+ CD133+CD29+ EPCs were obtained by using the Flow Cytometer (BD Bioscience, USA). The EPCs were subjected to 50 μg/ml LPS to establish LPS-induced cell injury models in EPCs.
Ficoll paque plus reagent
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom
Ficoll-Paque PLUS reagent is a density gradient medium used for the isolation of mononuclear cells from blood or bone marrow. It is a sterile, pyrogen-free solution composed of polysucrose and sodium diatrizoate.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Thp 1, by RIKEN BioResource Center (2 mentions)
Hl 60, by RIKEN BioResource Center (2 mentions)
Paxgene tube, by BD (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Chromium single cell 3 reagent kit v2, by 10x Genomics (1 mentions)
Single cell 3 chip kit, by 10x Genomics (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Tripure isolation reagent, by Roche (1 mentions)
Dnazol reagent, by Thermo Fisher Scientific (1 mentions)
Abi 3500 sequencer, by Thermo Fisher Scientific (1 mentions)
Seqman v 7, by DNASTAR (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
29 protocols using ficoll paque plus reagent
1
Isolation and Characterization of EPCs
2
Sero-Epidemiology of Convalescent COVID-19
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Patients were participants of the SERO-BL-COVID-19 study sponsored by the Department of Health, Canton Basel-Landschaft, Switzerland. All analyzed patients tested positive for SARS-CoV-2 after RT-PCR of naso- and oropharyngeal swab samples and experienced a resolution of COVID-19 symptoms without requiring hospitalization. Whole blood was collected 25 to 39 days following a positive RT-PCR test and subjected to density gradient centrifugation using the Ficoll Paque Plus reagent (GE Healthcare, #17-1440-02). After separation, the upper plasma layer was collected for semiquantitative ELISA detection of IgG and IgA SARS-CoV-2-specific antibodies (Euroimmun Medizinische Labordiagnostika, #EI2668-9601G, #EI2606-9601A). Peripheral blood mononuclear cells (PBMC) were collected from the interphase, resuspended in freezing medium (RPMI 1640, 10%(v/v) FBS, 10%(v/v) dimethyl sulfoxide) and cryopreserved in liquid nitrogen. Point-of-care lateral flow immunoassays assessing the presence of IgG and IgM SARS-CoV-2-specific antibodies (Qingdao Hightop Biotech, #H100) were performed at the time of blood collection. Both ELISA and point-of-care lateral flow tests employed Spike and Nuclear Capsid Protein for SARS-CoV-2-specific antibody detection.
3
PBMNC Isolation and MDSC Phenotyping
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Peripheral blood mononuclear cells (PBMNC) were isolated through Ficoll-paque PLUS reagent (GE Healthcare, Sweden) centrifugation and were analyzed within 6 hours (h) after collection.16 (link) The phenotype of MDSC was analyzed for the cell surface markers, including CD33, CD11b, human leukocyte antigen-D-related (HLA-DR), CD14, CD15 and lineage-specific markers (Lin), as described in the Online Supplementary Appendix. This marker combination allows the identification of MDSC (CD33+CD11b+HLA-DR-) and three MDSC subsets: PMN-MDSC (CD33+CD11b+HLA-DR-CD15+CD14-), M-MDSC (CD33+CD11b+HLA-DR-CD14+CD15-) and eMDSC (CD33+CD11b+HLA-DR-Lin-). Intracellular expression of Arg-1 and iNOS were also determined as described before.17 (link) All stained cells were detected by a FACS Canto II flow cytometer (BD Biosciences) and data were analyzed with FlowJo V10 (BD Biosciences).
4
Single-cell transcriptome analysis of PBMCs
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PBMCs were isolated from another blood samples of Dis6 and Con4 using the Ficoll-Paque Plus reagent (GE Healthcare). The PBMC suspensions were then loaded onto a haemocytometer for cell counting and viability examination. The viability of all the assessed samples exceeded 80%, and the cell densities of the suspensions were adjusted to 700–1200 cells/μL. The suspensions were then loaded onto microfluidic chips from the Single Cell 3′Chip Kit (10 × Genomics, CA, USA) to prepare single-cell gel beads-in-emulsions (GEMs). GEMs were subsequently subjected to cDNA library construction using a Chromium Single Cell 3′ Reagent Kit v2 (10 × Genomics). Libraries were sequenced using the BGISEQ-500 instrument. CellRanger software (version 5.0.1) was used to process the raw reads, map the reads, and quantify gene expression. Seurat software (version 3.0.2) was used for quality control and to identify highly variable genes. Single-cell RNA transcriptome analysis was then performed based on the expression matrices generated from the above steps.
5
Differentiation of Leukemia Cell Lines
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The THP-1 (human acute monocytic leukemia), HL-60 (human acute promyelocytic leukemia) and K562 (chronic myelogenous leukemia) cell lines were obtained from Bioresource Collection and Research Center (Hsinchu, Taiwan). Peripheral blood mononuclear cells (PBMCs) from one healthy volunteer donor were isolated using Ficoll-Paque Plus Reagent (GE Healthcare, Buckinghamshire, UK). The cells were maintained in RPMI-1640 medium (2 mM glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES and 1 mM sodium pyruvate) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc., Rockford, IL, USA) and 1% NEAA in a 5% CO2 incubator at 37 °C. For cell differentiation, the cells were seeded onto 6-well plates and treated with PMA (200 nM) or ATRA (10 μM for THP-1 and HL-60 cells or 8 μM for K562 cells) for 72 h.
6
HTLV-1 and ATL Cell Line Maintenance
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HTLV-1-transformed cell lines (MT-4, C8166, C91PL, and 1186.94 [31 (link)–33 (link)]) and ATL-derived cell lines, IL-2-independent (ED-40515(−), TL-Om1, and ATL-25 [34 (link), 35 (link)]), were maintained in RPMI-1640 media supplemented with penicillin, streptomycin, and 10% fetal bovine serum (FBS). ATL-derived cell lines, IL-2-dependent (LMY1, ATL-55T, ATL-43T SO4, KK1 [36 (link)–42 (link)]), were maintained in RPMI-1640 media supplemented with IL-2 (50 U/mL), penicillin, streptomycin, and 10% FBS. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by using Ficoll-Paque PLUS reagent (GE Healthcare Life Sciences). PBMCs were maintained in RPMI-1640 media supplemented with penicillin, streptomycin, and 20% fetal bovine serum (FBS). WRN inhibitor NSC 19630 was purchased from EMD Millipore’s Calbiochem® and NSC 617145 was purchased from Tocris Bioscience. Cells were treated with different concentrations of WRN inhibitors, and cells exposed to DMSO were used as a control, as indicated in figure legends.
7
Isolating PBMC and Extracting Nucleic Acids
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Peripheral blood mononuclear cells were isolated from blood with Ficoll®-Paque Plus reagent (GE Healthcare, Uppsala, Sweden) in accordance with the manufacturer’s instructions. Genomic DNA was extracted with the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and total RNA was extracted using TriPure isolation reagent (Roche, Indianapolis, IN), both in accordance with the manufacturer’s instructions. The DNA quality was confirmed by agarose gel electrophoresis. The concentrations of DNA and RNA were measured with a NanoDrop 2000 spectrophotometer.
8
Extracellular Vesicle Isolation from Plasma
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Polyethylene glycol (PEG, Fisher Scientific/ThermoFisher, P: 156–500, molecular weight = 8000 Da) was made at a stock concentration of 400 mg/mL in 1X PBS and stored at 4°C. PEG was added to the 50 g sample to a final concentration of 40 mg/mL. EV were precipitated at 4°C overnight and the precipitate collated by centrifugation at 1200 g for 60 min at 4°C. The pellet was resuspended in 375 µL of 1X PBS, yielding a combined (CFF + PEG) concentration factor for volume of ~ 800x (375 μL/300 mL).
For the isolation of EV from human plasma, blood was isolated from four healthy donors. Phase separation was done using Ficoll-Paque Plus Reagent (GE Healthcare, P: 17–1440-02). The upper layer (plasma) was then diluted in 1X PBS + 2 mM EDTA pH = 8.0 at a 1:1 ratio. The diluted plasma was then centrifuged at 12,000 g at 4°C for 15 min to pellet platelets, coagulation factors, larger macromolecules, etc. The soluble solution was then removed passed through a 0.22 µm Nalgene Rapid Flow Filter. EV were then processed through the AKTA Flux CFF and precipitated as described above. Final EV pellets were resuspended in 1X PBS + 1 mM EDTA for a concentration factor for volume of 100x.
For the isolation of EV from human plasma, blood was isolated from four healthy donors. Phase separation was done using Ficoll-Paque Plus Reagent (GE Healthcare, P: 17–1440-02). The upper layer (plasma) was then diluted in 1X PBS + 2 mM EDTA pH = 8.0 at a 1:1 ratio. The diluted plasma was then centrifuged at 12,000 g at 4°C for 15 min to pellet platelets, coagulation factors, larger macromolecules, etc. The soluble solution was then removed passed through a 0.22 µm Nalgene Rapid Flow Filter. EV were then processed through the AKTA Flux CFF and precipitated as described above. Final EV pellets were resuspended in 1X PBS + 1 mM EDTA for a concentration factor for volume of 100x.
9
PBMC Isolation from Whole Blood
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Early in the morning, fresh venous whole blood was collected from each participant in a 2.5 ml PAXgene tube (BD, Franklin Lakes, NJ, USA), followed by the isolation of PBMCs within 6 h of collection. PBMCs were isolated at room temperature (18-20 °C) using Ficoll-Paque PLUS reagent (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s instruction.
10
Isolation and Culture of Hematopoietic Cell Lines
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THP-1, HL-60, HEL 92.1.7, U-937, and K562 cell lines were obtained from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Peripheral blood mononuclear cells (PBMCs) were isolated and prepared from healthy volunteer donors using Ficoll-Paque Plus Reagent (GE Healthcare, Buckinghamshire, UK). These cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc., Rockford, IL, USA) and 1% NEAA in a 5% CO2 incubator at 37 °C.
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