4–6 week old NSG mice were irradiated with 0.8 Gy (X-ray) and injected i.v. with 2 × 105 human CD34+ cells, isolated from cord blood using CD34 Microbead Kit UltraPure (Miltenyi Biotec). Cord blood units were obtained from Anthony Nolan Cell Therapy Centre (ANCTC). On some occasions frozen CD34 + cells were used. Tail blood samples were taken to examine human immune cell reconstitution at 16–23 weeks post cell transfer. Two mice in which circulating conventional CD4+ T cells had very low CD45RA staining (<5%) were not used for experiments. Humanised mice were treated with control Ab, Abatacept, IgG-(IL-2)2 or both Abatacept and IgG-(IL-2)2. Treatment regimen: 600 μg Abatacept in 100 μl PBS was injected i.p. on d0, d3 and d6; 4 pmole (0.7 μg) IgG-(IL-2)2, developed by Roche35 (link), in 100 μl buffer (provided by Roche) was injected s.c. on d0, d4 and d6. At d7, spleen cells were harvested for analysis. Control Ab (human non-FcγR binding human IgG1) was also provided by Roche and injected at a 0.7 μg dose following the same injection schedule as for IgG-(IL-2)2.
Cd34 microbead kit ultrapure
Manufactured by Miltenyi Biotec
Sourced in Germany, United States, France
The CD34 MicroBead Kit UltraPure is a laboratory equipment product designed for the isolation and enrichment of CD34+ cells from various sample types. It utilizes magnetic beads coated with anti-CD34 antibodies to selectively bind and separate the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Stemspan sfem 2, by STEMCELL (6 mentions)
Ficoll paque, by GE Healthcare (3 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Cd71 microbead kit, by Miltenyi Biotec (2 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (2 mentions)
Cd34 microbeads kit, by Miltenyi Biotec (2 mentions)
Apc anti human cd34 clone 563, by BD (2 mentions)
Methocult opti h3404, by STEMCELL (2 mentions)
Magnetic activated cell sorting separation columns, by Miltenyi Biotec (2 mentions)
Fetal bovine serum (fbs), by STEMCELL (1 mentions)
Human bone marrow, by Lonza (1 mentions)
Stemvision, by STEMCELL (1 mentions)
Smartdish, by STEMCELL (1 mentions)
Accutase, by STEMCELL (1 mentions)
Methocult sf h4636, by STEMCELL (1 mentions)
Lineage cell depletion kit, by Miltenyi Biotec (1 mentions)
Tat cre recombinase, by Merck Group (1 mentions)
Recombinant human scf, by Miltenyi Biotec (1 mentions)
X vivo 10, by Lonza (1 mentions)
Flt3l, by Miltenyi Biotec (1 mentions)
50 protocols using cd34 microbead kit ultrapure
1
Humanized Mice for Immunotherapy Evaluation
2
HSC and MSC Co-culture CFU Assay
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Hematopoietic stem cells (HSCs) were isolated from human bone marrow (Lonza) using the CD34 MicroBead Kit UltraPure (Miltenyi Biotech, Bergisch Gladbach, North Rhine-Westphalia, Germany), frozen, and stored at −180°C. 1×103 HSCs and MSCs were resuspended in 0.1 mL Iscove’s Modified Dulbecco’s Medium, IMDM with 2% FBS (StemCell Technologies, Vancouver, BC, Canada). This HSC:MSC resuspension was added to 1 mL MethoCult H4034 Otimum (StemCell Technologies) and plated in a 6 well SmartDish (StemCell Technologies). CFU assays were quantified on day 14 using the STEMvision (StemCell Technologies) automated colony counter.
3
CD34+ Cell Isolation and CFU Assay
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Cells were dissociated into single cell solution using accutase (StemCell Technologies). CD34+ cells were purified from a portion of the cells using the CD34 Microbead Kit Ultrapure and LS columns (Miltenyi Biotec) according to the manufacturer’s instructions. Colony forming unit assays were performed using methylcellulose containing media (MethoCult H4434 Enriched and MethoCult SF H4636, StemCell Technologies) according to the manufacturer’s instructions. After 14 days in culture, cells were analyzed immunophenotypically via flow cytometry.
4
Isolation and Transduction of Hematopoietic Progenitor Cells
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Frozen umbilical cord blood cells were thawed and washed, and CD34+ progenitor cells were isolated using the CD34 MicroBead Kit UltraPure according to the manufacturer’s procedure (Miltenyi Biotec, 130-100-453). The magnetic separation was performed using 2 MACS columns (Miltenyi Biotec), consistently reaching a CD34+ purity of approximately 95%.
Lineage– progenitors from mouse BM were isolated using the Lineage Cell Depletion Kit (Miltenyi Biotec, 130-090-858). Tat-Cre recombinase (MilliporeSigma, SCR508) was added to 0.5 × 106 to 1.0 × 106 progenitor cells/mL in PBS at a concentration of 2 μM and incubated for 20 minutes at 37°C to recombine the floxed stop cassette in vitro. Cells were washed and incubated overnight in serum-free X-VIVO 10 (Lonza, BE04-743Q) supplemented with recombinant human SCF (50 ng/mL, Miltenyi Biotec), TPO (20 ng/mL, R&D Systems), and FLT3L (50 ng/mL, Miltenyi Biotec).
Lineage– progenitors from mouse BM were isolated using the Lineage Cell Depletion Kit (Miltenyi Biotec, 130-090-858). Tat-Cre recombinase (MilliporeSigma, SCR508) was added to 0.5 × 106 to 1.0 × 106 progenitor cells/mL in PBS at a concentration of 2 μM and incubated for 20 minutes at 37°C to recombine the floxed stop cassette in vitro. Cells were washed and incubated overnight in serum-free X-VIVO 10 (Lonza, BE04-743Q) supplemented with recombinant human SCF (50 ng/mL, Miltenyi Biotec), TPO (20 ng/mL, R&D Systems), and FLT3L (50 ng/mL, Miltenyi Biotec).
5
Isolation of Human CD34+ HSPCs
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Human fetal thymus and liver were obtained from Advanced Bioscience Resources (ABR). The UCLA institutional review board (IRB) determined these tissues are not human subjects and IRB review is not required since fetal tissues were obtained without patient identifying information from deceased fetuses. Human CD34+ HSPCs were isolated from fetal liver using magnetic beads conjugated with anti-hCD34+ monoclonal antibodies (CD34 MicroBead Kit UltraPure, human, Miltenyi Biotec) and an AutoMACS pro separator (Miltenyi Biotec). A human thymus from the same donor was used for generation of BLT mice.
6
Transcriptome Profiling of Allogeneic CD34+ Cells
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Four leukapheresis products from GCSF mobilized healthy allogeneic stem cell donors that were to be discarded otherwise were thawed, and PBMCs were isolated using Ficoll-Paque gradient centrifugation. Lineage positive cells were removed using the Lineage Depletion Kit (Miltenyi), and CD34+ cells were isolated from the lineage-depleted PBMCs using the CD34 Microbead Kit, Ultrapure (Miltenyi). Total RNA was purified using the RNeasy Plus Mini Kit (Qiagen). The sequencing libraries were prepared at the High Throughput Sequencing Facility (The University of North Carolina at Chapel Hill) using the TruSeq RNA Sample Prep Kit (Illumina), and pair-end sequencing (100 cycles/end) was performed on an Illumina HiSeq2000.
7
Differentiation of Primary Human Mast Cells
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Primary human mast cells were differentiated as described in65 (link). CD34+ hematopoietic progenitors were isolated from mobilised peripheral blood (Department of Transfusion & Tissue Medicine of the Brno University Hospital) using the CD34 MicroBead Kit UltraPure (Miltenyi Biotec) following manufacturer recommendation.
5 × 106 CD34+ cells were seeded at a concentration of 105 cells/ml in human mast cell media (StemPRO-34 (Gibco), 100 U/ml Penicillin, 100 ug/ml Streptomycin, 1 × GlutaMAX (Gibco), 100 ng/ml rhSCF (Peprotech), 100 ng/ml rhIL6 (Stemcell Technologies)). During the first week of culture, 100 ng/ml rhIL3 (Stemcell Technologies) were added to the media. Media was changed weekly by hemi-depletion and cells were maintained at a concentration below 5 × 105/ml.
Cells were used for experiments after 8 weeks of culture.
5 × 106 CD34+ cells were seeded at a concentration of 105 cells/ml in human mast cell media (StemPRO-34 (Gibco), 100 U/ml Penicillin, 100 ug/ml Streptomycin, 1 × GlutaMAX (Gibco), 100 ng/ml rhSCF (Peprotech), 100 ng/ml rhIL6 (Stemcell Technologies)). During the first week of culture, 100 ng/ml rhIL3 (Stemcell Technologies) were added to the media. Media was changed weekly by hemi-depletion and cells were maintained at a concentration below 5 × 105/ml.
Cells were used for experiments after 8 weeks of culture.
8
Cord Blood and Peripheral Blood CD34+ Cell Isolation
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Human nontherapeutic-grade CB samples were collected from informed and consenting mothers at the Marseille University Hospital de la Conception birth clinic, obtained through the Marseille CB bank and used for CD34+ cell enrichment (> 95%), as described (Balan et al., 2014 (link)), or from the UCB (USA national CB program). Alternatively, already purified CB CD34+ cell were purchased from a properly licensed commercial company (ABCell Bio). PBMCs were prepared by Ficoll (GE Healthcare) gradient centrifugation from buffy coats received from EFS (Marseille) or New York blood center (Long island city, NY, USA). Non-mobilized adult blood CD34+ cells were isolated using the CD34 MicroBead Kit UltraPure (Miltenyi). Donor cells were negative for HBV, HCV, HIV1/2, bacteria, fungi, and mycoplasma.
9
Optimized Expansion of CD34+ Cells
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CD34+-enriched cells from leukofilters (TACSI, Terumo BCT, Zaventem, Belgium) were expanded using a previously described two-phase optimized protocol11 (link). Briefly, the filter extract was enriched in CD34+ cells by magnetic activated cell sorting (CD34 MicroBead Kit UltraPure, Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were then seeded in StemSpan serum-free expansion medium (SFEM) supplemented with 20 µg/mL human low-density lipoprotein and a cocktail of cytokines (CC220, Stemcell Technologies, Vancouver, BC, Canada) and with 1 µM SR1 (Stemcell Technologies). On day 7, the cells were harvested, washed, seeded in StemSpan SFEM containing 1 µM SR1, 50 ng/mL TPO and 20 µg/mL human low-density lipoprotein and cultured for an additional 6 days. The cultures were incubated at 37 °C under normoxic conditions and a 5% CO2 atmosphere.
10
Isolation of Human CD34+ Cells
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Human CD34+ cells were collected from peripheral blood (PB) using a Ficoll-Hypaque density gradient. Briefly, blood sample was diluted 1:4 with Phosphate-Buffered Saline (PBS) with 2 mM EDTA and centrifuged at 800× g for 20 min at room temperature (RT) in a swinging-bucket rotor without brake. Following centrifugation, the mononuclear cell (MNC) ring was then recovered and washed twice with PBS. CD34+ were isolated through the immunomagnetic CD34+ positive selection using CD34 MicroBead Kit UltraPure (Miltenyi Biotec, Bergish Gladbach, Germany).
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