Female and male NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice from the Jackson Laboratories (Bar Harbor, ME, USA) or female Balb/c mice (BALB/cJRj, Janvier labs, Le Genest-Saint-Isle, France) were used for all experiments. Human CD34+ hematopoietic stem cells were isolated from human cord blood by magnetic separation (EasySep™ Human Cord Blood CD34 Positive Selection Kit II, STEMCELL technologies, Cologne, Germany) following the manufacturer’s protocol and purity controlled by flow cytometry. Humanized NSG (huNSG) mice were generated by engraftment of 100,000 hCD34+ cells (of a donor mix) at the age of 6 – 8 weeks, similar to the procedures described in earlier publications (15 (link)–18 (link)). Briefly, mice received whole-body irradiation with a sub-lethal dose of 2 Gy and hematopoietic stem cells were administered intravenously 2 hours later. For the generation of murinized NSG mice (muNSG), mice were treated as the huNSG mice, but received 100,000 bone marrow cells from a Balb/c donor instead of human CD34+ cells. The peripheral blood of huNSG mice was analyzed for the presence and frequency of murine CD45+, as well as human CD45+, CD3+ and CD20+ cells at week 18 post engraftment by flow cytometry.
Easysep human cord blood cd34 positive selection kit 2
Manufactured by STEMCELL
Sourced in Canada
The EasySep™ Human Cord Blood CD34 Positive Selection Kit II is a laboratory equipment product used to isolate and enrich CD34-positive cells from human cord blood samples. It utilizes magnetic particles and a specialized buffer system to selectively separate the target cells from the rest of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Lymphoprep, by STEMCELL (4 mentions)
Sepmate columns, by STEMCELL (3 mentions)
Facsaria cell sorter, by BD (3 mentions)
Retronectin coated plate, by Takara Bio (2 mentions)
Hil 6, by STEMCELL (2 mentions)
X tremegene transfection reagent, by Merck Group (2 mentions)
X mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Enzyme free dissociation buffer, by Thermo Fisher Scientific (2 mentions)
Amicon ultra centrifugal filter, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Balb cjrj, by Janvier Labs (1 mentions)
Nod cg prkdcscid il2rgtm1wjl szj mice, by Jackson ImmunoResearch (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Magnetic beads, by STEMCELL (1 mentions)
Methocult gf h4435 medium, by STEMCELL (1 mentions)
Sh800 cell sorter, by Sony (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
7 aminoactinomycin d, by Thermo Fisher Scientific (1 mentions)
Cd45ra fitc hi100, by BioLegend (1 mentions)
Cd38 pe cy7 hb 7, by BioLegend (1 mentions)
13 protocols using easysep human cord blood cd34 positive selection kit 2
1
Generation of Humanized and Murinized NSG Mice
2
MDSC Suppression Assay with Imprime
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Cord blood was purchased from National Disease Research Interchange (NDRI) and CD34+ stem cells were purified with EasySep™ Human cord blood CD34 positive selection kit II (StemCell Technologies). MDSCs were prepared by culturing CD34+ stem cells in RPMI medium supplemented with FLT-3L, GM-CSF, and G-CSF for 9 days. HLA-DR+ cells were then depleted from the differentiated cells with an anti-HLA-DR monoclonal Ab per vendor’s instruction (StemCell Tech). The HLA-DR-depleted MDSCs were treated with Imprime (25 μg/ml) or vehicle for 2 hours and then analyzed by flow cytometry and used in the MDSC suppression assay (20 (link)). For the MDSC suppression assay, PBMCs prepared from WB were labeled with CFSE, and 2.5 x 104 labeled PBMCs were added to the wells of a 96-well plate and incubated with MDSCs at the indicated PBMC : MDSC ratio in the presence of Dynabeads™ Human T-activator CD3/CD28 (Thermo Fisher Scientific). PBMC proliferation was measured after 3 days by flow cytometry.
3
Humanized Mouse Model Generation
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Human CD34+ cells were isolated from cord blood using EasySep Human Cord Blood CD34 Positive Selection Kit II (Stemcell Technologies) and were cryopreserved in IMDM containing 7.5% DMSO. For MISTRG-6-15 mice, 1–3 day-old newborn mice were humanized through injection of 1.5–3×104 cord blood CD34+ cells intrahepatically. For NSG mice, 1–3 day-old newborn mice preconditioned with sublethal irradiation (80 cGy) followed by intrahepatic injection of 1×105 cord blood CD34+ cells. Reconstitution of human CD45+ cells in blood was determined 9–10 weeks after engraftment. Mice were grouped after checking for blood engraftment to ensure that animals from different treatment groups or time points had similar levels of human T cells, NK cells, and macrophages. In each treatment group and at any time point, both male and female mice were used. Mice were randomly sorted into different treatment groups and time points. Identical cord blood donors were used when possible for experiments, but variation in donors between experiments does exist.
4
Production of MLL-AF9 Transduced Human CD34+ Cells
5
Isolation and Culture of Human Hematopoietic Stem Cells
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CD34+ cells from human umbilical CB were isolated via a two-step procedure using Ficoll-Plaque gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden) and EasySep Human Cord Blood CD34 Positive Selection Kit II (STEMCELL Technologies). For batch and single cell sorting of VAP-1+ and VAP-1− cells from CB we used a Sony SH800 cell sorter. This sorter applies low shear stress on cells allowing better survival during cell culture. CD34+ cells separated by magnetic beads (Stem Cell Technologies) were sorted into VAP-1+ and VAP-1− HSC using the following markers: Lineage−CD34+CD38−CD90+CD45RA−CD49f+. The sorted cells were then cultured as single cells in 96 well plates with Methocult GF + H4435 medium (Stem Cell Technologies). Single cells were cultured for 14 days, washed and replated as single colonies in 24 well plates for another 12 -14 days. Colonies were evaluated for CFUs by light microscopy.
6
Isolation and Transduction of Human CD34+ Cells
7
Isolation of Placental CD34+ Cells
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Figure 1 below shows the summary of the methods used in our study. After informed consent was obtained from the mothers, placentas and their UCB from healthy full term (FT) pregnancies were collected after delivery following protocols approved by the institutional review boards at Michigan State University, East Lansing and Sparrow Hospital, Lansing, MI, USA.
Mononuclear cells (MNCs) were isolated by density gradient centrifugation (Lymphoprep: STEMCELL Technologies, Vancouver, BC, Canada). After centrifugation, plasma samples were collected and immediately processed for exosomes isolation or stored in aliquots at −80 °C for later processing. MNCs were then aspirated and washed with PBS to remove excess Lymphoprep or plasma. CD34+ cells were isolated from MNCs using magnetic positive selection (EasySep™ Human Cord Blood CD34 Positive Selection Kit II: STEMCELL Technologies, Vancouver, BC, Canada) following the manufacturer’s instructions.
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Mononuclear cells (MNCs) were isolated by density gradient centrifugation (Lymphoprep: STEMCELL Technologies, Vancouver, BC, Canada). After centrifugation, plasma samples were collected and immediately processed for exosomes isolation or stored in aliquots at −80 °C for later processing. MNCs were then aspirated and washed with PBS to remove excess Lymphoprep or plasma. CD34+ cells were isolated from MNCs using magnetic positive selection (EasySep™ Human Cord Blood CD34 Positive Selection Kit II: STEMCELL Technologies, Vancouver, BC, Canada) following the manufacturer’s instructions.
8
Production of MLL-AF9 Transduced Human CD34+ Cells
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Production of MLL-AF9 transduced human CD34+ cells has been described.78 Human CD34+ cells were isolated from human cord blood (New York Blood Center) using the EasySep™ Human Cord Blood CD34 Positive Selection Kit II (Stemcell Technologies). For pre-enrichment, Lymphoprep (Stemcell Technologies) and SepMate™ columns (Stemcell Technologies) were used. Viral supernatants were generated by co-transfection of HEK293-T cells with retroviral (pMSCV-MLL-AF9-IRES-GFP)78 expression vector with packaging and envelope vectors (Human Retro: pUMVC and VSV-G) and X-tremeGene transfection reagent (Millipore). The viral supernatant was filtered through 0.45μm and was concentrated using Amicon Ultra centrifugal filters (Millipore). Human CD34+ cells were plated on retronectin-coated plates (Takara) and were spin infected at a 1:1 dilution of virus:media at 800g at 37°C for 1.5h. The cells were dissociated from the plates using enzyme-free dissociation buffer (Gibco) and were plated in fresh media. Cells were maintained in IMDM with 20% FBS, 1x penicillin/streptomycin (Gibco), 1xß-mercaptoethanol (Gibco), 6 μg/mL hIL3, 10 μg/mL hIL6, 10 μg/mL hSCF, 10 μg/mL TPO, and 10 μg/mL FLT3 (Stemcell Technologies) at 37°C and 5% CO2. Two days after transduction, GFP positive cells were sorted using FACSAria cell sorter (BD Bioscience).
9
Isolation of CD34+ Cord Blood Hematopoietic Stem Cells
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Deidentified umbilical cord blood (UCB) samples were obtained following scheduled cesarean section deliveries of healthy infants at the Department of Gynecology and Obstetrics, Aarhus University Hospital, Denmark. Consent was obtained from the mothers, but studies on anonymized samples, such as those used in the present study, are exempt from ethics permissions in Denmark (Kommiteeloven §§14. 3). CD34+ CB HSPCs (CB-HSPCs) were subsequently purified using EasySep Human Cord Blood CD34 Positive Selection kit II according to the manufacturer’s instructions (STEMCELL Technologies). Briefly, a pre-enrichment of CD34+ cells was performed where bi-specific antibodies targeting unwanted cells were used during standard Ficoll-Hypaque (GE Healthcare) density-gradient centrifugation. CD34+ cells were subsequently isolated using anti-CD34 immunomagnetic beads (positive selection). CD34+ CB HSPCs were either freshly used or cryopreserved until use.
10
Enrichment of Human Stem Cells
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Human stem cells, defined by expression of hCD34, were enriched from umbilical cord blood obtained during caesarean section births of females. Briefly, human stem cells were isolated using EasySep™ Human Cord Blood CD34 Positive Selection Kit II (Stemcell, Vancouver, Canada) column system. The isolated cells were above 85% CD34+ with less than 1% T cell contamination as determined by flow cytometry as previously shown [12 (link)] (S2 Fig ). Cells were cryopreserved in liquid nitrogen until transplantation.
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