An average of 2.5 ml of venous blood was collected into sodium heparin glass vacutainers from premature infants enrolled with consent in the Prematurity and Respiratory Outcomes Program (PROP), at the time of hospital discharge at the University of Rochester and the University at Buffalo and shipped to a central laboratory in Rochester. Freshly purified PBMCs were isolated by Ficoll gradient (Amersham Pharmacia Biotech # 17-1440-03) centrifugation, from the whole blood diluted 1:2 with 1x dPBS, and counted according to previously established protocols (39 (link)). In subjects with at least 8 million cells, 5 million cells were stained with antibodies to individual lymphocyte markers, and sorted on a FACSAriaII sorter at the Flow Cytometry Core facility of the University of Rochester as previously reported (16 (link)). CD3+CD8+, CD3+CD4+, CD3-CD56+ (NK), or CD3-CD19+ (B) cells were collected separately. Non-marker positive and dead cells were discarded. Sorted cells were spun into pellets, which were further lysed and frozen. The steps from collection to lysis of each sample were completed within a 24-h period in order to preserve RNA quality and integrity. Frozen lysates were thawed and RNA was extracted using Agilent Absolute RNA Microprep kit (catalog #400805), with an on-column DNase digestion, as per manufacturer recommended protocol.
Ficoll gradient
Manufactured by Cytiva
Sourced in United States
Ficoll gradient is a laboratory technique used for the separation and isolation of cells or other biological materials based on their density. It is a versatile tool that can be used to purify a variety of cell types, including lymphocytes, stem cells, and other mononuclear cells. The Ficoll gradient exploits differences in the density of various cellular components to achieve effective separation and isolation.
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Lab products found in correlation
Retronectin, by Takara Bio (2 mentions)
Absolute rna microprep kit, by Agilent Technologies (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Honeywell (1 mentions)
Rs4 11, by American Type Culture Collection (1 mentions)
Cd45 af700, by BioLegend (1 mentions)
Gr 1 apc, by BioLegend (1 mentions)
Cd11b pe cy7, by BioLegend (1 mentions)
Rbc lysis buffer, by BioLegend (1 mentions)
Ma900, by Sony (1 mentions)
X vivo 15 media, by Lonza (1 mentions)
Recombinant il 13 protein, by BioLegend (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Buffy coats, by Cytiva (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
10 protocols using ficoll gradient
1
Isolation and Characterization of Immune Cells from Preterm Infants
2
Isolation and Culture of Human PBMCs
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Human PBMCs were isolated from buffy coats of healthy donors using Ficoll gradient (Amersham, United Kingom) and cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 1% Penicillin-Streptomycin, non-nssential Amino Acids, L-glutamine, Sodium Pyruvate, HEPES and 10% heat-inactivated fetal bovine serum (Hyclone). Buffy coats from healthy donors were obtained from the Scripps Normal Blood donor program in La Jolla, California. Informed consent was obtained before all blood donations.
3
Cryopreservation of Bone Marrow Cells
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Patient and control samples obtained through bone marrow aspirate (5-10 mL) were collected with heparin (anticoagulant). The samples were rapidly prepared using a Ficoll gradient (1.077 g/mL) (Amersham Biosciences, Freiburg, Germany) and subsequent red blood cell lysis. Cells were then frozen in RPMI 1640 with 20% heat inactivated fetal bovine serum (Sigma, Saint Louis, MO, USA) and 5% DMSO (Riedel-de Haen, Seelze, Germany) in isopropanol-filled containers and subsequently stored in liquid nitrogen. When needed for analysis, cells were thawed and centrifuged to remove the supernatant and the pellet was used.
4
Cell Lines and PBMC Isolation Protocol
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Raji, Raji-2R, and Raji-4RH cells were generously provided by Matthew Barth, from Roswell Park Cancer Institute, Buffalo, New York, USA.11 (link) K526-mbIL21-41BBL cells were generously provided by Dean A. Lee, from Nationwide Children’s Hospital, Columbus, Ohio, USA.23 (link) Ramos, Daudi, and RS4;11 cells were purchased from ATCC. DOHH-2 cells were ordered from DSMZ. Raji, Raji-2R, Raji-4RH, and K526-mbIL21-41BBL cells were cultured in RPMI 1640 medium with 10% heat inactivated fetal bovine serum (FBS) at 37°C with 5% CO2. Obinutuzumab was generously provided by Christian Klein, from Roche, Zurich, Switzerland. N-820 and N-803 were generously provided by Hing Wong, Peter R. Rhode, John H. Lee, and Jeffrey T. Safrit, from ImmunityBio/Altor Bioscience, Culver City, California, USA. All fetal bovine serum used for cell culture and assays was heat-inactivated (heating to 56°C for 30 min) before use in order to inactivate complement. Leucocytes were obtained after informed consent from healthy donors at the New York Blood Center, New York, New York, USA. PBMCs were obtained by Ficoll gradient (Amersham Biosciences, Piscataway, New Jersey, USA) separation as we previously described.25 (link)
5
Establishing Cell Lines and Primary NK Cells for Immunotherapy Research
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U2OS (OS), M059K (GBM) and SKNFI (NB) cell lines were purchased from the American Type
Culture Collection, Gaithersburg, Maryland. K526-mbIL21-41BBL cells were generously
provided by Dean A. Lee, MD/PhD from Nationwide Children’s Hospital, Columbus,
Ohio.22 Dinutuximab was generously provided by United Therapeutics, Silver
Springs, Maryland. N-803 was generously provided by Hing Wong, PhD, Peter R. Rhode, PhD,
John H. Lee, MD, and Jeffrey T. Safrit, PhD from ImmunityBio/Altor Bioscience, Culver
City, California. Leukocytes were obtained after informed consent from healthy donors at
the New York Blood Center, New York, New York. Peripheral blood mononuclear cells (PBMNCs)
were obtained by Ficoll gradient (Amersham Biosciences, Piscataway, New Jersey, USA)
separation as we previously described.17 (link) U2OS,
M059K and SKNFI cells were cultured in DMEM medium supplemented with 10% fetal
bovine serum (FBS) and antibiotics penicillin and streptomycin (100 µg/mL).
K526-mbIL21-41BBL cells were cultured in complete medium (RPMI1640 medium supplemented
with 10% FBS and penicillin and streptomycin (100 µg/mL)). NK cells were
cultured in complete medium with 50 IU IL-2.
Culture Collection, Gaithersburg, Maryland. K526-mbIL21-41BBL cells were generously
provided by Dean A. Lee, MD/PhD from Nationwide Children’s Hospital, Columbus,
Ohio.22 Dinutuximab was generously provided by United Therapeutics, Silver
Springs, Maryland. N-803 was generously provided by Hing Wong, PhD, Peter R. Rhode, PhD,
John H. Lee, MD, and Jeffrey T. Safrit, PhD from ImmunityBio/Altor Bioscience, Culver
City, California. Leukocytes were obtained after informed consent from healthy donors at
the New York Blood Center, New York, New York. Peripheral blood mononuclear cells (PBMNCs)
were obtained by Ficoll gradient (Amersham Biosciences, Piscataway, New Jersey, USA)
separation as we previously described.17 (link) U2OS,
M059K and SKNFI cells were cultured in DMEM medium supplemented with 10% fetal
bovine serum (FBS) and antibiotics penicillin and streptomycin (100 µg/mL).
K526-mbIL21-41BBL cells were cultured in complete medium (RPMI1640 medium supplemented
with 10% FBS and penicillin and streptomycin (100 µg/mL)). NK cells were
cultured in complete medium with 50 IU IL-2.
6
Purification and Sorting of GFP+ Myeloid Cells
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HDC+ bone marrow cells were obtained from HdcGFP mice by crushing leg, arm, and pelvic bones in HBSS containing 2% heat-inactivated fetal bovine serum (900-108; Gemini). Single-cell suspensions were purified on a Ficoll gradient (17544202; Cytiva), and then red blood cells were lysed using 1× RBC lysis buffer (420301; BioLegend). The cells were stained with the following antibody cocktail: CD45-AF 700 (1:800, 103127; BioLegend), CD11b-PE-Cy7 (1:800, 101215; BioLegend), and Gr-1–APC (1:400, 108411; BioLegend). Propidium iodide was used as a viability dye to exclude dead cells. Cells were sorted using a Sony MA900 with a 100-micron nozzle. CD45+, CD11b+, Gr-1+, and GFP+ cells were sorted directly into X-VIVO 15 media (04-418Q; Lonza) for further culture experiments, or directly into TRIzol LS for RNA isolation.
7
Healthy Donor PBMC Isolation
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PBMCs from a 65-year-old individual (M) were used as a control for the flow cytometry experiments. Mononuclear cells were isolated from a commercially available healthy blood leukapheresis pack (New York Biologics Inc) over a Ficoll gradient (Cytiva, 17144003).
8
PBMC Isolation and Th2 Differentiation
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Blood samples from allergic asthma patients were collected and were isolated PBMCs using a Ficoll gradient (Cytiva, Marlborough, MA, USA). Isolated PBMCs were cultured in RPMI medium with recombinant human M-CSF (hM-CSF) protein (BioLegend) at 25 ng/ml for five days, and then, cultured with recombinant IL-13 protein (BioLegend) at 20 ng/ml for one day. Next, MSCs (1×104 cells) were treated for one day and harvested for analysis.
For Th2 cell differentiation, a cell plate was coated with 1 μg/ml CD3 Monoclonal Antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for one day. After coating, 1×105 PBMCs were seeded with 1 μg/ml CD28 Monoclonal Antibodies (Thermo Fisher Scientific) and stimulated with recombinant IL-2, IL-4, and IL-13 (10 ng/ml) for 3 days. Finally, following one day of MSC (1×104 cells) exposure, cells were harvested for analysis.
For Th2 cell differentiation, a cell plate was coated with 1 μg/ml CD3 Monoclonal Antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for one day. After coating, 1×105 PBMCs were seeded with 1 μg/ml CD28 Monoclonal Antibodies (Thermo Fisher Scientific) and stimulated with recombinant IL-2, IL-4, and IL-13 (10 ng/ml) for 3 days. Finally, following one day of MSC (1×104 cells) exposure, cells were harvested for analysis.
9
Lentiviral Transduction of Activated T Cells
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Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (San Diego Blood Bank) using Ficoll gradients (Amersham Biosciences). CD3+ T cells were isolated from PBMCs using the Pan T cell isolation kit (Miltenyi). For lentiviral transduction, cells were first activated for 72 hours using CD3/CD28-coated Dynabeads (Gibco) in complete RPMI medium with 100 IU/ml IL-2. The cells were then transduced with concentrated lentivirus cocktail at a multiplicity of infection of 10 for each virus by spinoculation on RetroNectin (Takara)–coated plates at 1800g, 32°C for 1 hour.
10
Culturing and Transducing Human Primary T Cells
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Human embryonic kidney cells (HEK 293T) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM of L-glutamine, 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 1 mM of sodium pyruvate at 37 °C with 5% CO2. All the cell culture reagents were purchased from Thermo Fisher Scientific. The plasmids were transfected into cells with Lipofectamine 3000 (Thermo Fisher Scientific, Cat. No. L3000015). Jurkat and P116 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin, 1% sodium pyruvate, and L-glutamine (300 mg/L). Human PBMCs were isolated from buffy coats (San Diego Blood Bank) after donor de-identification using Ficoll gradients (Amersham Biosciences). Human primary CD4+ T cells were isolated from PBMCs using CD4+ T cell isolation kit (Miltenyi). For lentiviral transduction, isolated CD4+ T cells were activated for 72 h in complete RPMI medium supplemented with phytohemagglutinin (PHA, Fisher Scientific, R30852801) and IL-2 (100 IU/mL). The cells were then infected with concentrated lentivirus at an MOI (multiplicity of infection) of 10 by spinoculation on Retronectin (Takara)–coated plates.
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