Easysep human cd34 positive selection kit 2

Manufactured by STEMCELL

Sourced in Canada

The EasySep Human CD34 Positive Selection Kit II is a laboratory product used to isolate and enrich CD34-positive cells from human samples. It utilizes magnetic particles and a specialized buffer system to selectively separate the target cells, without the need for columns or specialized equipment.

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Lab products found in correlation

Ficoll paque plus, by GE Healthcare (4 mentions) Ficoll paquetm plus, by GE Healthcare (3 mentions) Rbc buffer, by BioLegend (3 mentions) Histopaque 1077, by Merck Group (3 mentions) Ficoll paque density gradient, by STEMCELL (3 mentions) Rosettesep t cell depletion kit, by STEMCELL (2 mentions) G csf, by Thermo Fisher Scientific (2 mentions) Stemspan medium, by STEMCELL (2 mentions) Thrombopoietin, by Thermo Fisher Scientific (2 mentions) Stemspan sfem 2 medium, by STEMCELL (2 mentions) Rosettesep t cell enrichment cocktail, by STEMCELL (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by STEMCELL (1 mentions) Stemspan serum free expansion medium 2 sfem 2, by STEMCELL (1 mentions) Flt3l, by Thermo Fisher Scientific (1 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Stemspan cd34 expansion supplement, by STEMCELL (1 mentions) Stemspan, by Thermo Fisher Scientific (1 mentions) Hepes, by Merck Group (1 mentions)

28 protocols using easysep human cd34 positive selection kit 2

Isolation of CD34+ Cells from Cord Blood

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Cord blood samples were purchased from Anthony Nolan. Mononuclear cells (MNCs) were isolated from cord blood cells by centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences). CD34⁺ cell enrichment was performed using an EasySep Human CD34 Positive Selection Kit II (STEMCELL Technologies, cat. no. 17856) according to the manufacturer's instructions.

Hug N., Aitken S., Longman D., Raab M., Armes H., Mann A.R., Rio-Machin A., Fitzgibbon J., Rouault-Pierre K, & Cáceres J.F. (2022). A dual role for the RNA helicase DHX34 in NMD and pre-mRNA splicing and its function in hematopoietic differentiation. RNA, 28(9), 1224-1238.

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Expansion and Erythroid Differentiation of CD34+ Cells

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K562 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin/penicillin at 37°C in 5% carbon dioxide. Umbilical cord blood (CB) and adult peripheral blood (AB) samples were obtained from the Obstetrics Department of Beijing Maternity Hospital with informed consent for inclusion in the study signed by the donors. The mononuclear cells from CB and AB were isolated using lymphocyte separation solution Ficoll-Paque™ Plus (GE Healthcare, #17144002); then, primary CB and AB CD34⁺ cells were purified using the EasySep™ Human CD34 Positive Selection Kit II (STEMCELL, #17896, #17856) according to the manufacturer's instructions. Isolated CD34⁺ cells were cultured in StemSpan™ SFEM II medium for two phases [13 (link)]. Phase 1 involved cell proliferation and expansion for 7 days, and phase 2 involved erythroid differentiation for 16 days. The HUDEP-2 cell line donated by RIKEN Tsukuba Branch, Ibaraki, Japan, was cultured and differentiated as previously described [18 (link)].

Jin T., Zhang Z., Han Y., Li D., Liu J., Jiang M., Zhu J., Kurita R., Nakamura Y., Hu F., Xu Y., Fang X., Huang S, & Sun Z. (2022). Transmembrane Protein ANTXR1 Regulates γ-Globin Expression by Targeting the Wnt/β-Catenin Signaling Pathway. Journal of Immunology Research, 2022, 8440422.

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Isolation and Enrichment of CD34+ Cells

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Mononuclear cells (MNCs) were isolated from the bone marrow or cord blood cells by centrifugation using Ficoll-PaqueTM PLUS (GE Healthcare Life Sciences). CD34⁺ cell enrichment was performed using EasySep Human CD34 Positive Selection Kit II (StemCell Technologies) according to the manufacturer’s instructions.

Mian S.A., Philippe C., Maniati E., Protopapa P., Bergot T., Piganeau M., Nemkov T., Bella D.D., Morales V., Finch A.J., D’Alessandro A., Bianchi K., Wang J., Gallipoli P., Kordasti S., Kubasch A.S., Cross M., Platzbecker U., Wiseman D.H., Bonnet D., Bernard D.G., Gribben J.G, & Rouault-Pierre K. (2023). Vitamin B5 and succinyl-CoA improve ineffective erythropoiesis in SF3B1 mutated myelodysplasia. Science translational medicine, 15(685), eabn5135.

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Isolation of Human Hematopoietic Stem and T Cells

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Human bone marrow cells and G-CSF mobilized blood were collected from the remnants left-over in de-identified bags and filters used for clinical HSCT procedures. De-identified human umbilical cord blood samples were acquired from University of Colorado’s ClinImmune Labs cord blood bank or the Medical College of Wisconsin’s tissue bank. Venous peripheral blood was drawn from healthy consenting donors. Graft sources were isolated by ficoll density-gradient centrifugation (1100xg for 15min with 0 brake) to remove red blood cells, neutrophils and other non-leukocytes. Where indicated, StemCell Technologies RosetteSep T cell enrichment cocktail was used (catalog #15061) to remove lineage positive cells (excluding T cells), leaving untouched CD34⁺ HSPCs and T cells; T cells were depleted using StemCell Technologies RosetteSep T cell depletion kit (catalogue # 15661). HSPCs were isolated using StemCell Technologies EasySep Human CD34 positive selection kit II (catalog #17856) which first depletes lineage positive cells, followed by magnetic labeling of CD34⁺ cells and isolation using a magnetic column. Cells were counted, washed and resuspended in PBS prior to injection into mice.

Hess N.J., Hudson A.W., Hematti P, & Gumperz J.E. (2020). Early T cell Activation Metrics Predict GVHD In A Humanized Mouse Model of Hematopoietic Stem Cell Transplantation. Journal of immunology (Baltimore, Md. : 1950), 205(1), 272-281.

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Expansion and Electroporation of CD34+ Cells

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CD34 + cells were isolated from mononuclear cells derived from mobilized peripheral blood stem cells (PBSC) autologous transplant products by using EasySep Human CD34 Positive Selection Kit II (StemCell Technologies, 18056). Cell were cultured for 48 h before electroporation in StemSpan™ Serum-Free Expansion Medium II (SFEM II) (StemCell Technologies, 09605) with streptomycin (20 mg/mL), penicillin (20 unit/mL) and the following human cytokines (all from GenScript unless stated otherwise, catalog numbers and dilution used in parentheses): FLT3L (Z02926, 100 ng/mL), G-CSF (Z02980, 10 ng/mL), SCF (Z02692, 100 ng/mL), TPO (Pepro-Tech, 300-18, 25 ng/mL) and IL-6 (Z03034, 10 ng/mL). Cells were cultured at a density of 2.5*10^5 cells/ml in 96-well U-bottom plates.

Feldman T., Bercovich A., Moskovitz Y., Chapal-Ilani N., Mitchell A., Medeiros J.J., Biezuner T., Kaushansky N., Minden M.D., Gupta V., Milyavsky M., Livneh Z., Tanay A, & Shlush L.I. (2021). Recurrent deletions in clonal hematopoiesis are driven by microhomology-mediated end joining. Nature Communications, 12, 2455.

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In vitro Expansion of Mouse and Human Hematopoietic Stem Cells

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Sorted mouse Lin⁻c-Kit⁺ cells, LSK cells, and HSCs were cultured in vitro in Stem Span medium (Stemcell Technologies). For drug treatment, 5 µM RGFP-966 or 500 nM GNE-049 was added into the cultured medium for indicated times.
Human BM CD34⁺ cells were isolated with the EasySep Human CD34 Positive Selection Kit II according to the manufacturer’s instructions (Stemcell Technologies). The purity of CD34⁺ cells was >90%, as determined by flow cytometry. Isolated CD34⁺ cells were cultured in vitro in Stem Span medium (Stemcell Technologies) with Stem Span CD34⁺ Expansion Supplement (Stemcell Technologies). The use of human cells was approved by the institutional review board of Third Military Medical University.

Chen Z., Huo D., Li L., Liu Z., Li Z., Xu S., Huang Y., Wu W., Zhou C., Liu Y., Kuang M., Wu F., Li H., Qian P., Song G., Wu X., Chen J, & Hou Y. (2021). Nuclear DEK preserves hematopoietic stem cells potential via NCoR1/HDAC3-Akt1/2-mTOR axis. The Journal of Experimental Medicine, 218(5), e20201974.

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CD34+ Cell Isolation and Transduction

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Mononuclear cells (MNCs) were isolated from UCB or BM by centrifugation using Ficoll‐Paque PLUS (GE Healthcare Life Sciences) followed by red blood cell lysis. CD34⁺ cell selection was performed using the EasySep™ Human CD34 Positive Selection Kit II (StemCell Technologies) according to the manufacturer's instructions. CD34⁺ cells were stimulated using StemSpan medium (StemCell Technologies) supplemented with cytokines (150 ng/ml stem cell factor [SCF], 150 ng/ml FMS‐like tyrosine kinase‐3 ligand [Flt3‐L], 10 ng/ml interleukin 6, 25 ng/ml granulocyte colony‐stimulating factor, 20 ng/ml thrombopoietin [TPO]; PeproTech) and 1% hydroxyethylpiperazine‐ethane‐sulphonic acid buffer (HEPES; Sigma‐Aldrich) for 4–6 h. Virus particles were added to the stimulated cells at a multiplicity of infection (MOI) of 30 and incubated at 37°C, 5% CO₂ overnight. Cells were washed and re‐suspended in expansion medium (StemSpan with 150 ng/ml SCF, 150 ng/ml FlT3‐ligand, 20 ng/ml TPO; PeproTech) and expanded for 4 days prior to fluorescence‐activated cell sorting.

Armes H., Bewicke‐Copley F., Rio‐Machin A., Di Bella D., Philippe C., Wozniak A., Tummala H., Wang J., Ezponda T., Prosper F., Dokal I., Vulliamy T., Kilpivaara O., Wartiovaara‐Kautto U., Fitzgibbon J, & Rouault‐Pierre K. (2022). Germline ERCC excision repair 6 like 2 ( ERCC6L2 ) mutations lead to impaired erythropoiesis and reshaping of the bone marrow microenvironment. British Journal of Haematology, 199(5), 754-764.

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Isolation and Purification of CD34+ Cells

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MNCs from UCB and AML PB samples were isolated by density centrifugation using Ficoll‐Paque TM PLUS (GE Healthcare Life Sciences). Following on, red blood cell lysis was performed using ammonium chloride solution by incubating the cells at 4°C for 10 min.
CD34⁺ cell enrichment for UCB samples was performed using EasySep Human CD34 Positive Selection Kit II (StemCell Technologies) according to the manufacturer's instructions. For UCB experiments, individual donors were pooled together based on the sex of the donors.

Mian S.A., Ariza‐McNaughton L., Anjos‐Afonso F., Guring R., Jackson S., Kizilors A., Gribben J, & Bonnet D. (2024). Influence of donor–recipient sex on engraftment of normal and leukemia stem cells in xenotransplantation. HemaSphere, 8(5), e80.

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Isolation of CD34+ cells from SLE BM

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Human heparinized BM aspirate (10 ml) was collected from healthy and SLE patients and subjected to density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich). Briefly, blood was diluted 1:3 PBS and carefully layered over Histopaque medium. Tubes were centrifuged at 500 g for 30 min (no break) at room temperature. BM mononuclear cells were isolated using Histopaque-1077 (Sigma-Aldrich). BM mononuclear cells were washed, and erythrocytes were lysed with RBC buffer (420301, BioLegend). CD34⁺ cells were isolated using EasySep™ Human CD34 Positive Selection Kit II (18056, StemCell Technologies). Purity was tested and was >95%.

Filia A., Mitroulis I., Loukogiannaki C., Grigoriou M., Banos A., Sentis G., Giannouli S., Karali V., Athanasiadis E., Kokkinopoulos I, & Boumpas D.T. (2024). Single-cell transcriptomic analysis of hematopoietic progenitor cells from patients with systemic lupus erythematosus reveals interferon-inducible reprogramming in early progenitors. Frontiers in Immunology, 15, 1383358.

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Isolation and Purification of CD34+ HSPCs

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CD34⁺ HSPCs from various stages of human development were isolated by positive magnetic selection using the EasySep Human CD34 Positive Selection Kit II (Stem Cell Technologies) after mononuclear cell isolation on a Ficoll-Paque density gradient (Stem Cell Technologies). Purity of isolated cells was assessed by flow cytometry with a PE conjugated anti-human CD34 antibody (Supplementary Table 3). Freshly isolated CD34⁺ HSPCs were either immediately cultured or cryopreserved for later use.

Basak A., Munschauer M., Lareau C.A., Montbleau K.E., Ulirsch J.C., Hartigan C.R., Schenone M., Lian J., Wang Y., Huang Y., Wu X., Gehrke L., Rice C.M., An X., Christou H.A., Mohandas N., Carr S.A., Chen J.J., Orkin S.H., Lander E.S, & Sankaran V.G. (2020). Control of human hemoglobin switching by LIN28B-mediated regulation of BCL11A translation. Nature genetics, 52(2), 138-145.

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