As previously described [15 (link)], umbilical cord blood (UCB) was collected from umbilical veins after neonatal delivery with maternal informed consent. UCB harvests were processed within 24 h of collection. UCB was separated by isolating mononuclear cells with Ficoll-Hypaque solution (density, 1.077g/cm3; Sigma, St. Louis, MO, USA). The separated mononuclear cells were washed, suspended in Minimum Essential Medium a modification (MEM a; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), and seeded at a concentration of 5 x 105 cells/cm2. Cultures were maintained at 37°C in a humidified atmosphere (21% O2) containing 5% CO2 with a change of culture medium twice a week. One batch of cells was used to inject all pups. As described previously [16 (link)], human UCB derived MSCs from a single donor at passage 6 manufactured and also used for various preclinical studies of neonatal intractable disorders including BPD [17 (link)], IVH [18 (link)] and HIE [19 (link)] were obtained from Medipost Co., Ltd. (Seoul, Korea).
Ficoll hypaque solution
Manufactured by Merck Group
Sourced in United States
Ficoll-Hypaque solution is a density gradient medium used for the isolation of cells, such as mononuclear cells, from whole blood or other biological samples. It allows the separation of different cell types based on their density during centrifugation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Mem a, by Thermo Fisher Scientific (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ficoll hypaque, by Merck Group (1 mentions)
Dmem lg medium, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Collagenase 2, by Merck Group (1 mentions)
α minimum essential medium, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Merck Group (1 mentions)
27 protocols using ficoll hypaque solution
1
Umbilical Cord Blood-Derived Mesenchymal Stem Cell Protocol
2
Isolation of Rat Bone Marrow CD144+ ECs
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To promote the angiogenesis capacity of Alg + Fib hydrogel, bone marrow CD144+ ECs were isolated from rat long bones using magnetic activated cell sorting (MACS). After the induction of euthanization using an overdose of ketamine and xylazine, femurs and tibias were exposed and placed in DPBS. Under sterile conditions, the muscles and adipose tissue remnants were excluded. The extremities were cut using sterile scissors and bone marrow content was aspirated into the 10 cm culture plates (SPL, South Korea) using syringes with 20 gauge needles. In the next step, mononuclear cells were purified by gradient density centrifugation using Ficoll-Hypaque solution (Cat no: F5415; Sigma-Aldrich). In brief, samples were diluted with DPBS at a ratio of 1: 1 and gently overlaid to the same volume of Ficoll-Hypaque solution. The samples were centrifuged at 300–400 g for 30 min and cells at the interphase were isolated and washed twice with DPBS. Freshly isolated cells were blocked with 1% bovine serum albumin (Cat no: A2153; Sigma-Aldrich) incubated with anti-CD144 microbeads (Order no: 130-123-932; Miltenyi Biotech; Germany) at 4 °C for 1 h. Using LS columns (Order no; 130-042-401; Miltenyi Biotech; Germany) and magnetic stands, CD144+ ECs were enriched and collected in separate tubes.
3
Isolation of Rabbit Bone Marrow-Derived MNCs
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This study has been permitted by the Ethics Committee, Tabriz University of Medical Sciences (IR.TBZMED.VCR.REC.1397.132). At present study, two matured white rabbits (New Zealand race) were preserved in separate cages. After animal anaesthetization (35 mg/kg bw ketamine/5 mg/kg bw xylazine), the bone marrow blood content of femur was aspirated by Jamshidi needle containing 1000 IU/ml sodium heparin. Following dilution of obtained bloods with (volume ratio 1:1), MNCs were isolated using Ficoll-Hypaque protocol according to our previous study.21 (link) To this end, the prepared bloods were gradually overlaid on the Ficoll-Hypaque solution (Sigma-Aldrich) and centrifuged at 450g for 25 min. Afterwards, layer containing MNCs was gathered at between the plasma and the Ficoll solution. After washing with PBS, MNCs suspended in DMEM/LG medium (Gibco), supplemented with %5 FBS (Gibco). The exhausted culture mediums were changed every 4 days (Figure 1 ).
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This study has been permitted by the Ethics Committee, Tabriz University of Medical Sciences (IR.TBZMED.VCR.REC.1397.132). At present study, two matured white rabbits (New Zealand race) were preserved in separate cages. After animal anaesthetization (35 mg/kg bw ketamine/5 mg/kg bw xylazine), the bone marrow blood content of femur was aspirated by Jamshidi needle containing 1000 IU/ml sodium heparin. Following dilution of obtained bloods with (volume ratio 1:1), MNCs were isolated using Ficoll-Hypaque protocol according to our previous study.21 (link) To this end, the prepared bloods were gradually overlaid on the Ficoll-Hypaque solution (Sigma-Aldrich) and centrifuged at 450g for 25 min. Afterwards, layer containing MNCs was gathered at between the plasma and the Ficoll solution. After washing with PBS, MNCs suspended in DMEM/LG medium (Gibco), supplemented with %5 FBS (Gibco). The exhausted culture mediums were changed every 4 days (
4
Isolation of Peripheral Blood Lymphocytes
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Lymphocytes were isolated according to the procedure that had been described by Erniati et al. [30 (link)], with modifications in the number of blood samples. Peripheral blood samples (30 mL) were aseptically collected from a healthy adult human female with the approval of the IPB University Ethical Committee. The blood was immediately transferred into sterile tubes and centrifuged at 300 × g at 20°C for 10 min. The buffy coat layer obtained was passed through a Ficoll-Hypaque solution (Sigma-Aldrich) and centrifuged at 900 × g at 20°C for 30 min to obtain a lymphocyte ring. The cells were aspired carefully, PBS (Sigma-Aldrich) was added, and the solution was centrifuged again to obtain lymphocyte cell pellets. These were washed with PBS, and the cell viability was calculated by adding 10 μL of trypan blue to 10 μL of the cell suspension followed by counting with a hematocytometer under a microscope. The cells were further diluted with RPMI-1640 medium (Sigma-Aldrich) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich) to obtain a density of 1 × 106 cells/mL. The cell suspensions used to determine SI had viability ≥ 95%.
5
Intracerebral Transplantation of hUCB-MSCs for MCAO
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This study was approved by the Institutional Review Board of MEDIPOST Co., Ltd (IRB No. 201002-01) and hUCB-MSCs were separated and maintained as described previously [18 (link)]. Umbilical cord blood was collected from umbilical veins after neonatal delivery with informed consent of the pregnant mothers. hUCB-MSCs were isolated by separation of mononuclear cells (MNCs) using a Ficoll-Hypaque solution (d=1.077 g/cm3; Sigma). After transferring into minimum essential medium (MEM; Gibco) supplemented with fetal bovine serum (FBS; Gibco), MNCs were seeded in a culture flask at 5×105 cells/cm2. Cells maintained in a humidified 5% CO2 at 37℃ formed colonies of spindle-shaped cells. At 50% confluence, cells were harvested after treatment with 0.25% (w/v) trypsin-EDTA (Gibco) and were reseeded for expansion. Prepared cells were administrated into three experimental groups at the anteroposterior, +1.0 mm from bregma, laterally +3.0 mm and dorsoventrally -5.0 mm, according to the stereotaxic atlas for Paxino and Watson (1986): In group one, rats were given 1X PBS injected intrastriatally two days after MCAO. In group two, rats were given hUCB-MSCs (5.0×105 cells) in 5 µl total volume (1X PBS) injected intrastriatally two days after MCAO. In group three, rats were given hUCB-MSCs (5.0×105 cells) in 5 µl total volume (1X PBS) injected intrastriatally two and nine days after MCAO.
6
Isolation of Mononuclear Cells from Blood
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Mononuclear cells are known to express various TLRs, and thus served as positive control for the flow cytometry and PCR analysis. The cells were separated from outdated venous blood samples obtained from the Hadassah Medical Center Blood Bank. Blood samples were placed in 50 ml polypropylene tubes and mixed with equal volumes of PBS. Ficoll-Hypaque solution (Sigma Chemical Co. St. Louis) was slowly layered under the blood/PBS mixture. Afterwards, the 50 ml tube was centrifuged 30 min at 2000 rpm (900 g) with no brake. The mononuclear cell layer was aspirated using a sterile pipette and transferred into a second centrifuge tube.
7
Neonatal Umbilical Cord Blood Mesenchymal Stem Cell Spheroid
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Neonatal UCB samples were collected from the umbilical vein after obtaining informed consent from the 5 different donors. This protocol was approved by the Institutional Review Board of MEDIPOST Co. Ltd. (MP-2014-04). The UCB samples were isolated by separating mononuclear cells (MNCs) using a Ficoll-Hypaque solution (Sigma, St. Louis, MO, USA). Separated mononuclear cells were suspended in a minimum essential medium (a-MEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 50 μg/mL gentamicin (Gibco, Carlsbad, CA, USA), respectively. The cells were cultured and incubated at 37 °C in a humidified atmosphere containing 5% CO2. The culture medium was replaced with fresh medium twice a week, as previously described [34 (link)]. Neutralizing antibody treatment was performed to block E-cadherin expression. After trypsinization of the monolayered UCB-MSCs, as described above, they were then cultured in hanging drops that contained 20 μL of spheroid medium containing 100,000 cells/drop with E-cadherin neutralizing antibody (Sigma, St. Louis, MO, USA) or immunoglobulin G (R&D Systems, Minneapolis, MN, USA). The spheroid was incubated at 37 °C for 72 h. UCB-MSCs used in all experiments were at passage 6.
8
Culturing HepG2 Cells and PBMCs
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HepG2 cells, derived from a liver hepatocellular carcinoma of a 15-year-old Caucasian male, were purchased from ATCC. HepG2 cells were grown in a polystyrene flask in complete culture medium consisting of EMEM (Eagle’s minimum essential medium, 1.2 g/L sodium bicarbonate, non-essential amino acid, L-glutamine and sodium pyruvate) supplemented with 10% FBS and PenStrep (100 U/mL penicillin, and 100 µg/mL streptomycin). Cells were cultured in a humidified 5% CO2 incubator at 37 °C.
Peripheral blood mononuclear samples were obtained from healthy donors from the City of Hope National Medical Center. PBMCs were isolated from whole blood by centrifugation in SepMate™ tubes through a Ficoll-Hypaque solution (Histopaque-1077, Sigma-Aldrich). following manufactured protocol. Suspension cells were cultured in RPMI 1610 with 10% v/v FBS, PenStrep and 100 U/mL interleukin 2. Cells were cultured in a humidified 5% CO2 incubator at 37 °C.
Peripheral blood mononuclear samples were obtained from healthy donors from the City of Hope National Medical Center. PBMCs were isolated from whole blood by centrifugation in SepMate™ tubes through a Ficoll-Hypaque solution (Histopaque-1077, Sigma-Aldrich). following manufactured protocol. Suspension cells were cultured in RPMI 1610 with 10% v/v FBS, PenStrep and 100 U/mL interleukin 2. Cells were cultured in a humidified 5% CO2 incubator at 37 °C.
9
Isolation and Expansion of hUCB-MSCs
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hUCB-MSCs were provided by MEDIPOST Co., Ltd. (Seoul, Korea). This study was approved by the Institutional Review Boards of the Seoul National University Hospital and MEDIPOST Co., Ltd. Umbilical cord blood was collected from the umbilical veins after neonatal delivery with informed consent of the pregnant mother. MSCs were isolated from mononuclear cells (MNCs) using a Ficoll-Hypaque solution (Sigma). Following transfer to α-minimum essential medium (α-MEM; Gibco) supplemented with fetal bovine serum (FBS; Gibco), MSCs were seeded in culture flasks at 5×105 cells/cm2. Cells maintained in humidified 5% CO2 at 37℃ formed colonies of spindle-shaped cells. At 50% confluence, cells were harvested after treatment with 0.25% trypsin-EDTA (Gibco) and were reseeded for expansion.
10
Isolation and Expansion of hUCB-MSCs
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hUCB-MSCs were collected from the umbilical cord vein of a newborn baby, with the consent of the mother. To isolate and expand MSCs from cord blood, mononuclear cells (MNCs) were isolated using a Ficoll–Hypaque solution (d = 1.077 g/cm3; Sigma). The cells were then seeded at 5 × 105 cells/cm2 in culture flasks. After colonies of spindle-shaped cells were formed, the cells were reseeded for expansion. hUCB-MSCs were cultured in αMEM medium supplemented with 10% FBS and gentamycin in a humidified atmosphere containing 5% CO2 and various concentrations of oxygen (3 or 21%) at 37 °C.
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