Human CD34 + cells were enriched from umbilical cord blood samples and the mothers were tested negative for HIV, HBV, HCV and Treponema pallidum. Informed consent was obtained from each mother and the study was approved by the Institutional Ethics Review Board of the 1st Affiliated Hospital of Nanchang University. Cord blood was separated on a Ficoll-Hypaque plus density gradient (GE healthcare) and umbilical cord blood mononuclear cells (UCBMC) were isolated. The isolated UCBMCs were then subjected to magnetic separation to enrich CD34 + cells (Miltenyi Biotec, Cat. No.: 130–046-702).
Cd34 cells
Manufactured by Miltenyi Biotec
Sourced in Germany
CD34+ cells are a population of hematopoietic stem and progenitor cells that express the CD34 antigen on their surface. These cells are commonly used in research and clinical applications related to stem cell biology, hematopoiesis, and regenerative medicine.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll density gradient separation, by Miltenyi Biotec (2 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Ficoll hypaque plus density gradient, by GE Healthcare (1 mentions)
Magnetic bead selection, by Miltenyi Biotec (1 mentions)
Nsg mice, by Jackson ImmunoResearch (1 mentions)
Human scf, by Thermo Fisher Scientific (1 mentions)
48 well plate, by BD (1 mentions)
Human gm csf, by Thermo Fisher Scientific (1 mentions)
Human g csf, by Thermo Fisher Scientific (1 mentions)
Human m csf, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
7 protocols using cd34 cells
1
Isolation and Enrichment of CD34+ Cells from Umbilical Cord Blood
2
Generation of Humanized BLT Mice
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BLT hu-mice were generated as described previously25 ,28 (link),37 (link)–39 (link). Briefly, human fetal liver and thymus tissues (ca. 1-mm3, Advanced Bioscience Resources, Alameda, CA) were implanted under the kidney capsule of female NSG mice (6- to 8-week-old, Jackson Laboratories, Ellsworth, ME). The mice were bred at The Scripps Research Institute, and each cohort was produced using tissues from a single donor. Magnetic bead selection (Miltenyi Biotec, San Diego, CA) was used to isolate CD34+ HSPCs from autologous fetal liver tissue. The purified CD34+ cells (Miltenyi Biotec, San Diego, CA) were phenotyped cytometrically25 ,28 (link),37 (link)–39 (link), and cryopreserved until injection (200,000–350,000 CD34+ cells) into mice 3 weeks after Thy/Liv implantation. Human reconstitution in peripheral blood was verified by flow cytometry using previously described methods25 ,28 (link),37 (link)–39 (link), and the degree of humanization was determined using a gating strategy described elsewhere40 (link). Mice with an average > 65% of human CD45+ cells were selected to ensure successful HIV-1 infection.
3
Characterizing CALR Mutant HSCs
4
Generation of MDSCs from CD34+ Cells
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Human CB was provided from the Catholic Hematopoietic Stem Cell Bank after written informed consent given by normal full-term pregnant women. For MDSCs generation, isolated CD34+ cells (Miltenyi Biotec, Bergisch Gladbach, Germany) were cultured in a 48-well plate (BD Falcon, Bedford, MA) at 1 × 105 cells/ well with 1 ml of IMDM containing 10% FBS (Gibco, Grand Island, NY, United States), 10% penicillin–streptomycin (100 U/ml; Lonza Walkersville, MD, United States), 2 mM L-glutamine (Lonza Walkersville) (10% complete medium), 100 ng/ml human GM-CSF (300–03, PeproTech, Rocky Hill, NJ, United States), 100 ng/ml human G-CSF (300–23, PeproTech), or 100 ng/ml human M-CSF (300–25, PeproTech) and 50 ng/ml human SCF (300–07, PeproTech). After incubation for 7 days, the cells were removed from the 48 well plate and centrifuged at 1,300 rpm for 5 min. After one wash with serum free IMDM, the cells were cultured for 2 weeks and media was changed every 7 days. From weeks 4–6, the cells were cultured at a higher density (5 × 105 cells/well). Media was changed every 7 days throughout 6 weeks of the culture.
5
Isolation of Human CD34+ Cells
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Human CD34+ cells were isolated from umbilical cord blood donated by healthy volunteers at the department of gynecology and obstetrics, The First Affiliated Hospital of Nanchang University. The volunteers were tested negative for HIV, hepatitis B virus, hepatitis C virus, and Treponema pallidum. Informed consent was obtained from each volunteer, and the study was approved by the Institutional Ethics Review Board of The First Affiliated Hospital of Nanchang University. Umbilical cord blood mononuclear cells (UCBMCs) were isolated from cord blood on a Ficoll-Paque Plus density gradient (GE Healthcare, Chicago, Illinois, USA). The isolated UCBMCs were then subjected to magnetic separation to isolate CD34+ cells (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat. No.: 130-046-702).
6
Isolation and Culture of HEK293T and PBMCs
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HEK293T cells (human embryonic kidney cells; ATCC) were expanded and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% penicillin/streptomycin (Merck Millipore, Billerica, MA, USA) at 37°C with 5% CO2. PBMCs were isolated by Ficoll gradient centrifugation as described previously.13 (link) Immune magnetic separation was used to isolate CD14+ and CD34+ cells (Miltenyi Biotec, Bergisch-Gladbach, Germany) as described.13 (link)
7
Characterizing CALR Mutant HSCs
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Peripheral blood from three ET patients with mutations in CALR underwent Ficoll density gradient separation, immunomagnetic selection for CD34+ cells (Miltenyi Biotech), and fluorescence-activated cell sorting (FACS) (Influx, Becton-Dickinson) using PeCy7-labeled CD34, clone 561 (lot# B257238, BioLegend), APC-labeled CD38, clone HIT2 (lot #B247250, BioLegend) and FITC-labeled CD10, clone HI10a (lot# B254556, BioLegend) antibodies were used to isolate CD34+CD38−, CD34+CD38+, and CD34+CD10+ cell compartments. DNA was extracted from sorted cells (Qiagen) and the VAF of CALR mutations was measured by droplet digital PCR (QX200 Droplet Digital PCR System, Bio-Rad) with primers that specifically detect CALR type 1 mutations (52-bp deletion (p.L367fs*46), CALR type 2 mutations (5-bp TTGTC insertion (p.K385fs*47) or wildtype alleles.
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