Cd34 microbead

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

CD34 microbeads are magnetic beads coated with antibodies that specifically bind to the CD34 protein expressed on the surface of hematopoietic stem and progenitor cells. They are designed for the isolation and enrichment of CD34-positive cells from a variety of cell sources.

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Lab products found in correlation

Clinimacs device, by Miltenyi Biotec (3 mentions) Ls column, by Miltenyi Biotec (3 mentions) Ficoll paque, by GE Healthcare (2 mentions) Quadromacs separator, by Miltenyi Biotec (2 mentions) Facsaria 3, by BD (2 mentions) Mirneasy mini kit, by Qiagen (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Accutase, by Thermo Fisher Scientific (2 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (2 mentions) Interleukin 7 (il 7), by Thermo Fisher Scientific (2 mentions) Il 15, by Thermo Fisher Scientific (2 mentions) Pf 06651600, by Pfizer (2 mentions) Tofacitinib, by Merck Group (2 mentions) Fcr blocking reagent, by Miltenyi Biotec (2 mentions) Affymetrix genechip human genome u133 plus 2.0 array, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Anti cd8 antibody, by BioLegend (1 mentions) Anti cd4 antibody, by BioLegend (1 mentions) Anti cd34 antibody, by BioLegend (1 mentions) Anti cd3 antibody, by BioLegend (1 mentions)

Close spelling variants of this product

CD34 MicroBead Kit (230 citations) Human CD34 MicroBead Kit (73 citations) CD34 MicroBead Kit UltraPure (50 citations) Anti-CD34 microbeads (23 citations) CD34 MicroBead Kit UltraPure human (14 citations) Indirect CD34 MicroBead Kit (8 citations) CD34 Microbead Isolation Kit (6 citations) MACS CD34 MicroBead Kit Ultra Pure (4 citations) Direct CD34+ MicroBead kit (3 citations) Miltenyi CD34 Microbead kit (3 citations)

53 protocols using cd34 microbead

Haploidentical Hematopoietic Progenitor Cell Graft Preparation

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HPCs were obtained via G-CSF mobilization of the haploidentical donor, and collection by leukapheresis on day 5 and 6 of G-CSF. The first HPC product collected on day 5 was T-cell-depleted using the CliniMACS device and CD34 Microbead (Miltenyi Biotec, Auburn, CA, USA). Minimum cell dose required for the CD34+ enriched progenitor cell graft was 2 × 10⁶ CD34+ cells/kg. Maximum CD3+ dose allowed for the CD34+ enriched HPC was 0.1 × 10⁶ CD3+ cells/kg.
The HPC product collected on day 6 was processed for CD45RA+ cell depletion using the CliniMACS device and its “Depletion 3.1” software. There was no target CD34+ dose or CD3+ dose on the CD45RA+ depleted product; however release criteria of the product includes a ≥2 log₁₀ depletion of CD45RA+ cells. Two of the 17 patients did not meet the minimum CD45RA depletion after a single depletion step. Per standard operating procedure, a second run under the same conditions was performed and the requisite level of depletion was achieved in both products.
The NK cell product was collected by leukapheresis 5 days after the second HPC collection. It was a nonmobilized product, and was processed on the CliniMACS device as previously described.^{28 (link), 29 (link)} All three cell products were infused fresh.

Triplett B.M., Shook D.R., Eldridge P., Li Y., Kang G., Dallas M., Hartford C., Srinivasan A., Chan W.K., Suwannasaen D., Inaba H., Pui C.H, & Leung W. (2015). Rapid Memory T-cell Reconstitution Recapitulating CD45RA-depleted Haploidentical Transplant Graft Content in Patients with Hematologic Malignancies. Bone marrow transplantation, 50(7), 968-977.

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Haploidentical Hematopoietic Progenitor Cell Graft Preparation

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Mobilization and Enrichment of Hematopoietic Stem Cells

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Donor peripheral blood stem cells were mobilized by administering granulocyte colony-stimulating factor (10 μg/kg per day for 4 days) and were harvested on day +5 by 1 to 2 leukapheresis procedures. The first hematopoietic progenitor cell product was T cell-depleted using the CliniMACS device and CD34 Microbead (Miltenyi Biotec, Bergisch Gladbach, Germany). The minimum cell dose required for the CD34 + -enriched progenitor cell graft was 4 × 10 6 CD34 + cells/kg. The maximum CD3 + dose allowed for the CD34 + -enriched hematopoietic progenitor cell graft was .1 × 10 5 CD3 + cells/kg. The CD34 -fraction was processed for CD45RA + cell depletion using the CliniMACS device and its Depletion 3.1 software (Miltenyi Biotec) . The maximum CD3 + CD45RA + dose allowed was .1 × 10 5 CD3 + CD45RA + cells/kg and a ≥2.5 log 10 depletion of CD45RA + cells. The second fraction was infused after the CD34 + fraction, on the same day or the day after. Both cell products were infused fresh.

Sisinni L., Gasior M., de Paz R., Querol S., Bueno D., Fernández L., Marsal J., Sastre A., Gimeno R., Alonso L., Badell I., López-Granados E., Torres J., Medina L., Torrent M., Diaz de Heredia C., Escudero A, & Pérez-Martínez A. (2018). Unexpected High Incidence of Human Herpesvirus-6 Encephalitis after Naive T Cell-Depleted Graft of Haploidentical Stem Cell Transplantation in Pediatric Patients. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 24(11).

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PBMC Isolation and CAR-T Cell Generation

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Density-gradient centrifugation was used to isolate PBMCs from healthy donor samples or patients with MM by Ficoll-Paque (General Electric). T cells were isolated from PBMCs with anti-CD3 microbeads (Miltenyi Biotec, 130-050-101) and were cultured in AIM V Medium (Thermo Fisher Scientific) with 5% human AB serum (MilliporeSigma) and 400 IU IL-2 (R&D Systems). Dynabeads of human T-activator CD3/CD28 (Thermo Fisher Scientific, 11161D) were added for T cell expansion and activation (26 (link)). T cells were activated for 2–3 days prior to transduction.
Lentivirus particles were used to transduce T cells. T cells and concentrated lentiviruses were added into RetroNectin-precoated plates (Takara Bio) (26 (link)). Cells were cultured in AIM V Media for 24 hours, and the transduction step was repeated. After 24 hours, cells were washed with PBS and cultured in fresh media for 7 days. CAR-T cells were detected by BD FACSVerse flow cytometry with anti-CD34 antibodies (BioLegend, clone: 561, no. 343606), anti-CD3 antibodies (BioLegend, clone: HIT3a, no. 300318), and CD34 microbeads (Miltenyi Biotec, 130-046-703) for isolation. CD4 or CD8 phenotypes of CAR-T cells were detected by flow cytometry using anti-CD4 antibodies (BioLegend, clone: RPA-T4, no. 300508) and anti-CD8 antibodies (BioLegend, clone: SK1, no. 344722).

Sun F., Cheng Y., Chen J.R., Wanchai V., Mery D.E., Xu H., Gai D., Al Hadidi S., Schinke C., Thanendrarajan S., Zangari M., van Rhee F., Tricot G., Shaughnessy JD J.r, & Zhan F. (2024). BCMA- and CST6-specific CAR T cells lyse multiple myeloma cells and suppress murine osteolytic lesions. The Journal of Clinical Investigation, 134(1), e171396.

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Isolation of AML and Healthy BM Cells

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This study was approved by the Committee for the Ethical Review of Research, Fujian Medical University Union Hospital (2018KY061) and informed consent was obtained from all the bone marrow providers. Human BM aspirates were collected from AML patients prior to treatment at Fujian Medical University Union Hospital, Fuzhou, China. Details on AML patient samples are shown in Table 1. AML blasts were purified using CD34 microbeads (Miltenyi Biotec, Shanghai, China). CD34⁺ hematopoietic stem cells (HSCs) were obtained from BM of healthy donors and were purified using CD34 microbeads.

You R., Hou D., Wang B., Liu J., Wang X., Xiao Q., Pan Z., Li D., Feng X., Kang L., Chen P, & Huang H. (2021). Bone marrow microenvironment drives AML cell OXPHOS addiction and AMPK inhibition to resist chemotherapy. Journal of Leukocyte Biology, 112(2), 299-311.

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Isolation of CD34+ Hematopoietic Stem Cells

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Fresh umbilical cord blood and adult peripheral blood were obtained from consenting donors by Shandong Cord Blood Hematopoietic Stem Cell Bank, while bone marrow from healthy donors and aplastic anemia patients were obtained from Blood Biobank of Institute of Hematology and Blood Diseases Hospital (please refer to “Reporting summary” for details). Histopaque®-1077 (Sigma-Aldrich) was used to separate mononuclear cells. CD34⁺ cells were isolated using CD34 MicroBeads and a QuadroMACS Separator (Miltenyi Biotec) according to the manufacturer’s instructions. Unless specified, each independent experiment used a mixture of no less than three units of umbilical cord blood as initial fresh cells for subsequent experiments to reduce individual variability.

Li Y., He M., Zhang W., Liu W., Xu H., Yang M., Zhang H., Liang H., Li W., Wu Z., Fu W., Xu S., Liu X., Fan S., Zhou L., Wang C., Zhang L., Li Y., Gu J., Yin J., Zhang Y., Xia Y., Mao X., Cheng T., Shi J., Du Y, & Gao Y. (2023). Expansion of human megakaryocyte-biased hematopoietic stem cells by biomimetic Microniche. Nature Communications, 14, 2207.

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Lentiviral Transduction of CD34+ Cells

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Cord blood units were obtained from the MD Anderson Cancer Center Cord Blood Bank under a protocol approved by the Institutional Review Board. Mononuclear cells were isolated from multiple units by density separation using Ficoll-Paque PREMIUM^® (GE Healthcare, Pittsburgh, PA) and pooled together. CD34⁺ cells were isolated from mononuclear cells by using CD34 microbeads (Miltenyi Biotec, San Diego, CA). For generating lentiviruses, 293T cells were co-transfected with viral expression and packaging plasmids. At 48 h thereafter, culture supernatants were harvested. Viruses were concentrated using PEG-it^® Virus Precipitation Solution (System Biosciences). CD34⁺ cells were pre-stimulated overnight in QBSF-60^® serum-free medium (Quality Biological, Gaithersburg, MD) supplemented with 100 ng/ml SCF, 100 ng/ml FLT3L and 100 ng/ml TPO, and then infected with lentiviruses using TransDux^® reagent (System Biosciences). At 24 h thereafter, cells were infected a second time and then incubated in QBSF-60^® serum-free medium supplemented with 50 ng/ml SCF, 80 ng/ml FLT3L, 50 ng/ml TPO and 100 ng/ml IL-6.

Trinh B.Q., Barengo N., Kim S.B., Lee J.S., Zweidler-McKay P.A, & Naora H. (2015). The homeobox gene DLX4 regulates erythro-megakaryocytic differentiation by stimulating IL-1β and NF-κB signaling. Journal of Cell Science, 128(16), 3055-3067.

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Differentiation and Activation of Human Dendritic Cells from Hematopoietic Stem and Progenitor Cells

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All recombinant cytokines were purchased from PeproTech (PeproTech, NJ). HSPCs were purified from G-CSF mobilized human PB using CD34⁺ microbeads (Miltenyi, 130046702) in accordance with the manufacturer’s instructions. To induce DCs, HSPCs were first cultured in Roswell Park Memorial Institute (RPMI) 1640 containing 10% fetal bovine serum (Dongling Biotech), FMS-like tyrosine kinase 3 ligand (FLT3L) (100 ng/mL), stem cell factor (SCF) (20 ng/mL), interleukin-3 (IL-3) (20 ng/mL), and thermoplastic polyolefin (TPO) (20 ng/mL) for 7 days, followed by additional culture for 7 days following the removal of TPO. On day 14, the cells were collected for analysis. To test the impact of inflammatory mediators [e.g., IFN-γ (10ng/ml), IL-1β (10ng/ml), G-CSF (10ng/ml) and TNF-α (10ng/ml)] and LPS (10ng/ml) on DC development, we cultured HSPCs as described above with or without addition of the inflammatory cytokines and the inducer as described above. To induce DC activation, we added LPS (100 ng/mL, Sigma), R848 (100 ng/mL, Invitrogen) or CpG oligonucleotide (CPG ODN) (1 μM) into the DC population or fluorescence-activated cell sorting (FACS)-sorted purified DC population using a BD Influx or BD FACs Aria II.

Lu J., Sun K., Yang H., Fan D., Huang H., Hong Y., Wu S., Zhou H., Fang F., Li Y., Meng L., Huang J, & Bai Z. (2021). Sepsis Inflammation Impairs the Generation of Functional Dendritic Cells by Targeting Their Progenitors. Frontiers in Immunology, 12, 732612.

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Transcriptomic Profiling of CAR T-Cells

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The dFab_CCR-IL2 positive and nontransduced human T cells from 3 individual donors were isolated using CD34 microbeads (Miltenyi Biotec, 130–046–702) and rested for 24 hours in R10 media. Subsequently, the T cells were seeded at 1 × 10⁶ cells/mL with or without 100,000 iU/mL of human IL2. After 72 hours, rHuIL2 was replenished. After 7 days, the cells were lysed, and the mRNA was extracted with the illustra RNAspin Mini RNA Isolation Kit (GE Healthcare, 12183018A). The RNA was quantified with NanoDrop (Thermo Fisher Scientific). The quality of the RNA was assessed with Agilent TapeStation (Agilent, RRID:SCR_019547). Poly-A selection was performed to remove ribosomal RNA. Roughly, 40 million paired end reads (150 bp) were sequenced on Illumina Hiseq sequencer (GeneWiz) for each individual CAR T-cell sample with different CCR modules.
Fastq reads were mapped to the human genome (B37.3) and gene model GenCode (V19) with Omicsoft Aligner 4 (18 (link)). Gene count data was normalized by logGenometric mean values. Differential gene expression analysis was carried out using DeSeq2 General Linear Model test (RRID:SCR_015687; ref. 19 (link)). Gene set enrichment analysis (Subramanian) Pre-ranked tool (GSEAPreranked) was used to conduct the pathway and functional analysis on the differentially expressed genes.

Righi M., Gannon I., Robson M., Srivastava S., Kokalaki E., Grothier T., Nannini F., Allen C., Bai Y.V., Sillibourne J., Cordoba S., Thomas S, & Pule M. (2023). Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors. Cancer Immunology Research, 11(9), 1203-1221.

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CD34+ Cell Enrichment and Cryopreservation

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In order to enrich samples in the progenitor and primitive fraction, we performed a positive selection using CD34 MicroBeads (Miltenyi Biotech), according to the manufacturer’s instructions. The CD34⁺ fraction was immediately used for functional assays, or cryopreserved for later use in freezing medium (DMEM 50%/HSA 40%/DMSO 10%). When available, 10,000 cells were used for purity assessment by flow cytometry.

Ruiz M.S., Sanchez M.B., Gutiérrez L., Koile D., Yankilevich P., Mosqueira C., Cranco S., Custidiano M.D., Freitas J., Foncuberta C., Moiraghi B., Pavlovsky C., Pérez M.A., Ventriglia V., Ávalos J.S., Mordoh J., Larripa I, & Bianchini M. (2018). Evaluation of the primitive fraction by functional in vitro assays at the RNA and DNA level represents a novel tool for complementing molecular monitoring in chronic myeloid leukemia. Oncotarget, 9(29), 20255-20264.

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