Venous blood from participants were collected in acid‐citrate dextrose‐containing vacutainers (BD Bioscience, San Jose, CA) and processed within 24 hours. Peripheral blood mononuclear cells (PBMCs), including CTCs, were separated from venous blood by gradient centrifugation with the use of Ficoll‐Paque solution (Sigma‐Aldrich, St. Louis, MO) using the manufacturer’s protocol.
Ficoll paque solution
Manufactured by Merck Group
Sourced in United States
Ficoll-Paque solution is a density gradient medium used for the isolation and purification of cells, organelles, and other biological particles. It is a sterile, pyrogen-free, and endotoxin-tested solution that facilitates the separation of different cell types based on their density.
Automatically generated - may contain errors
Lab products found in correlation
Acid citrate dextrose containing vacutainers, by BD (3 mentions)
Ebm 2, by Lonza (2 mentions)
Antibiotic antimycotic, by Merck Group (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Sds polyacrylamide gel electrophoresis sds page, by Merck Group (1 mentions)
Propidium iodide, by US Biological (1 mentions)
Rabbit polyclonal anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
Acid citrate dextrose vacutainer tube, by BD (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Omega Scientific (1 mentions)
10 protocols using ficoll paque solution
1
PBMC Isolation from Venous Blood
2
Isolation and Expansion of Umbilical Cord-Derived MSCs
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Human umbilical cords were obtained and processed within 24 h after delivery of
neonates. All procedures were approved by the Ethics Committee of Xi'an Jiaotong
University (China). Umbilical cord blood samples were diluted 1:1 in
phosphate-buffered saline (PBS) and mixed with 3% gelatin to deplete red blood cells.
The plasma fraction was collected and centrifuged at 2500 g for 5
min, and the cellular pellet was resuspended in alpha-minimum essential medium
(α-MEM). The cell suspension was transferred to centrifuge tubes containing twice the
volume of Ficoll-Paque solution (Sigma, USA) at a density of 1.077 g/mL, and
subjected to centrifugation at 2500 g for 20 min to isolate the
fraction of mononuclear cells that contained MSCs (17 (link)). The isolated cells were washed twice with D-Hank's buffer and
cultured at a density of 1×106 cells/cm2 in α-MEM containing
20% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Sigma). The relatively
high plating density facilitates rapid growth and expansion and assists cell
survival. The culture medium was changed every 2 days, and cells were subcultured
when they reached about 50% confluence.
neonates. All procedures were approved by the Ethics Committee of Xi'an Jiaotong
University (China). Umbilical cord blood samples were diluted 1:1 in
phosphate-buffered saline (PBS) and mixed with 3% gelatin to deplete red blood cells.
The plasma fraction was collected and centrifuged at 2500 g for 5
min, and the cellular pellet was resuspended in alpha-minimum essential medium
(α-MEM). The cell suspension was transferred to centrifuge tubes containing twice the
volume of Ficoll-Paque solution (Sigma, USA) at a density of 1.077 g/mL, and
subjected to centrifugation at 2500 g for 20 min to isolate the
fraction of mononuclear cells that contained MSCs (17 (link)). The isolated cells were washed twice with D-Hank's buffer and
cultured at a density of 1×106 cells/cm2 in α-MEM containing
20% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Sigma). The relatively
high plating density facilitates rapid growth and expansion and assists cell
survival. The culture medium was changed every 2 days, and cells were subcultured
when they reached about 50% confluence.
3
Cytotoxic Effects of Curcumin on Leukemia Cells
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PEG, HPMA, curcumin, acetone, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), Ficoll-Paque solution, Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), potassium chloride (KCl), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT), RIPA buffer (containing 50 mM Tris-HCl, 150 mM sodium chloride (NaCl), 1% Triton X-100, 0.5 mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate, and protease inhibitor cocktail), SDS-polyacrylamide gel electrophoresis (SD-SPAGE), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Sigma-Aldrich (St. Louis, MO, USA). K562, a potent leukemia cell line derived from chronic myelogenous leukemia patients in blast crisis, was purchased from the RIKEN BRC Cell Bank (Ibaraki, Japan). RPMI 1640 medium, fetal bovine serum, penicillin, and streptomycin were from Invitrogen™ Life (Carlsbad, CA, USA). Propidium iodide (PI) was from US Biological (Swampscott, MA, USA). Goat anti-rabbit IgG conjugated with HRP was from the Promega Corporation, (Madison, WI, USA). LuminataTM Forte Western HRP Substrate was from the Millipore Corporation, (Billerica, MA, USA). Primary rabbit polyclonal anti-WT1 and rabbit polyclonal anti-GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other chemicals and solvents were of the highest grade available.
4
Dog Oncology PBMC Isolation Protocol
5
Blood Sampling and PBMC Isolation
6
Isolation of Rat Bone Marrow Mononuclear Cells
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Ten Sprague-Dawley (SD) male rats weighing 150–180 g, provided by the Centers for Disease Control of Hubei Province, were anesthetized with 10% chloral hydrate and then killed. Fresh bone marrow was harvested aseptically by flushing the tibias and femurs with PBS. Mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque solution (d = 1.077, Sigma, USA), then seeded on T25 plates and cultured in EC basal medium-2 (EBM-2, Lonza, Switzerland) in a humidified atmosphere of 5% CO2 at 37°C. Seventy-two hours later, the unattached cells were washed away with media, and the adherent cells were cultured further with changed media.
7
Isolation of Peripheral Blood Neutrophils
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Peripheral blood neutrophils were separated using density gradient centrifugation as detailed in previous studies [25 (link),32 (link)]. Briefly, blood was collected through venipuncture in an acid-citrate-dextrose Vacutainer tube (BD Biosciences; Franlin Lakes, NJ, USA). This was followed by separation of polymorphonuclear neutrophils (PMNs) from the whole blood using Ficoll-Paque solution (1.077 g/mL; Sigma-Aldrich, St. Louis, MO, USA) as part of the density gradient centrifugation process. This process separates blood leukocytes into different bands, with PMNs staying at the bottom layer of the tube. Next, the PMN layer was processed using the dextran sedimentation method to isolate neutrophils with greater than 95% purity, which were subsequently utilized for different molecular analyses.
8
Isolation of c-kit+ Mononuclear Cells
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Ten rats were anesthetized using a surplus dose of Ketamine and Xylazine. After cervical dislocation, the extremities of the femurs were cut, and medullary content was extracted by pushing PBS containing 2% fetal bovine serum (FBS; Gibco). Mononuclear cells (MNCs) were isolated using gradient centrifugation Ficoll-Paque ® solution (Sigma-Aldrich). The samples were centrifuged at 400 g for 20 min, and monolayer cells were collected and washed twice for 5 minutes. The cells were incubated in PBS (1% FBS) at 4 °C for 30 min. The supernatant was removed, and the cells were incubated with anti-c-Kit microbeads according to the manufacturer's instructions. At the end of the procedure, c-kit + and c-kit -cells were isolated by passing through the LS columns (Miltenyi Biotec, Germany) [25] (link).
9
Isolation and Culture of Endothelial Progenitor Cells
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4-week-old male Sprague-Dawley (SD) rats provided by the Centers for Disease Control of Hubei Province were anesthetized with 3% pentobarbital sodium at 30 mg/kg and then killed, isolated the tibia and femur of the rats aseptically, and rinsed their bone marrow cavities with PBS buffer repeatedly to collect fresh bone marrow. Mononuclear cells were isolated by density gradient centrifugation using the Ficoll-Paque solution (d = 1.077, Sigma, USA). Then, they were inoculated in a T25 culture flask with endothelial cells basal medium-2 (EBM-2, Lonza, Switzerland) and placed in a cell incubator with 5% CO2 atmosphere at 37°C. Changing the media every 72 hours and the growth state of the cells were observed every day. In our previous experiments [27 (link)], we identified these cells by the property of uptaking acetylated LDL (ac-LDL) and Ulex europaeus agglutinin-1 (UEA-1) by EPCs, and cells cultured by this method have been identified as EPCs.
10
Isolation and Cryopreservation of PBMCs
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Venous blood was collected in acid-citrate dextrose-containing vacutainers (BD Bioscience, CA, USA) and stored at 4°C. All the blood samples were processed within 4 hours upon collection. PBMCs including CTCs were separated by gradient centrifugation with the use of Ficoll-Paque solution (Sigma-Aldrich, MO, USA) using the manufacturer's protocol. PBMCs were suspended in fetal bovine serum (FBS, Omega Scientific, CA, USA) and Dimethyl sulfoxide (DMSO, Fisher Scientific, NH, USA). Specimens were aliquoted into labeled cryovials at 1-mL whole blood equivalency and banked under -180°C at CSMC-UOPBBB. At time of experimentation, 2-mL whole blood equivalency of samples was retrieved from UOPBBB, immediately thawed completely in 37°C water bath. After washing by PBS, the PBMCs were re-suspended in 200-μL PBS for the TR-NanoVelcro CTC purification system.
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