概述

使用EasySep™人脐带血CD34阳性选择试剂盒II，通过免疫磁阳性选择从新鲜的全脐带血样本中分离高纯度的人CD34+细胞。EasySep™在已发表的研究中广泛使用了20多年，它结合了单克隆抗体的特异性和无柱磁系统的简单性。

在EasySep™阳性选择程序中，首先使用RosetteSep™人脐带血CD34预富集鸡尾酒（15896C）对造血祖细胞进行预富集，其中含有识别T细胞、B细胞、骨髓细胞和血小板表面标记物的抗体。然后使用EasySep™Human CD34 Positive Selection Cocktail （18096C）选择所需的CD34+细胞，其中包含识别CD34的抗体。本试剂盒中的鸡尾酒还含有一种针对人Fc受体的抗体，以防止非特异性结合。使用EasySep™磁铁和简单地倾倒或移液除去不需要的细胞来分离标记的细胞。感兴趣的细胞留在试管中。磁性细胞分离后，所需的CD34+细胞已准备好用于下游应用。

如果将CD34+细胞从新鲜血液或灰褐色皮毛中分离出来人全血CD34+细胞试剂盒（目录#15086）建议。

如果从其他样本中分离CD34+细胞，包括新鲜或先前冷冻的动员外周血或骨髓单个核细胞，或先前冷冻的脐带血单个核细胞，我们建议使用EasySep™人CD34阳性选择试剂盒II（目录#17856）.

该产品取代了独立的EasySep™人脐带血CD34阳性选择试剂盒（目录#18096）。

了解更多关于免疫磁性的知识EasySep™技术工作原理或如何完全自动化免疫磁细胞分离RoboSep™。探索额外的产品针对您的工作流程进行了优化，包括培养基、补充剂、抗体等。

Magnet Compatibility

• EasySep™ Magnet (Catalog #18000)

• “The Big Easy” EasySep™ Magnet (Catalog #18001)

• EasyEights™ EasySep™ Magnet (Catalog #18103)

• RoboSep™-S (Catalog #21000)

Subtype
Cell Isolation Kits

Cell Type
Hematopoietic Stem and Progenitor Cells

Species
Human

Sample Source
Cord Blood

Selection Method
Positive

Application
Cell Isolation

Brand
EasySep, RoboSep, RosetteSep

Area of Interest
Immunology, Stem Cell Biology

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Data Figures

Figure 1. Typical EasySep™ Human Cord Blood CD34 Positive Selection Profile

Starting with fresh cord blood, the CD34+ cell content of the isolated fraction is typically 91 ± 9% (mean ± SD) using the purple EasySep™ Magnet.

Figure 2. Isolation of CD34+ Cells Using EasySep™ Human Cord Blood CD34 Positive Selection Kit II

CD45 and CD34 expression of cells before separation (“Start”), after RosetteSep™ (“Pre-Enriched”), and after selection of CD34+ cells (“Isolated”) using EasySep™ Human Cord Blood CD34 Positive Selection Kit II (Catalog #17896). Results of a typical experiment are shown. CD45+CD34+ HSPCs (top right quadrant) have been enriched > 15-fold (from 0.6% to 10%) after RosetteSep™ pre-enrichment and > 200-fold (from 0.6% to 98%) after EasySep™ CD34+ selection (“Isolated”). The flow cytometry data shown are gated on cells with intermediate-to-high forward light scatter (FSC) that are negative for propidium iodide (PI) staining to exclude debris, RBCs, platelets, and dead cells. Based on the results of cell separations with 15 different CB samples with a starting CD34+ cell purity of 0.4%, the average CD34+ cell purity is 6% after RosetteSep™ pre-enrichment and 91% after EasySep™ cell isolation.

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type

Product Name

Catalog #

Lot #

Language

Document Type

Product Information Sheet

Product Name

RoboSep™ Human CB CD34 Positive Selection Kit II

Catalog #

17896RF

Lot #

All

Language

English

Document Type

Product Information Sheet

Product Name

EasySep™ Human Cord Blood CD34 Positive Selection Kit II

Catalog #

17896

Lot #

All

Language

English

Document Type

Safety Data Sheet 1

Product Name

RoboSep™ Human CB CD34 Positive Selection Kit II

Catalog #

17896RF

Lot #

All

Language

English

Document Type

Safety Data Sheet 2

Product Name

RoboSep™ Human CB CD34 Positive Selection Kit II

Catalog #

17896RF

Lot #

All

Language

English

Document Type

Safety Data Sheet 3

Product Name

RoboSep™ Human CB CD34 Positive Selection Kit II

Catalog #

17896RF

Lot #

All

Language

English

Document Type

Safety Data Sheet 4

Product Name

RoboSep™ Human CB CD34 Positive Selection Kit II

Catalog #

17896RF

Lot #

All

Language

English

Document Type

Safety Data Sheet 1

Product Name

EasySep™ Human Cord Blood CD34 Positive Selection Kit II

Catalog #

17896

Lot #

All

Language

English

Document Type

Safety Data Sheet 2

Product Name

EasySep™ Human Cord Blood CD34 Positive Selection Kit II

Catalog #

17896

Lot #

All

Language

English

Document Type

Safety Data Sheet 3

Product Name

EasySep™ Human Cord Blood CD34 Positive Selection Kit II

Catalog #

17896

Lot #

All

Language

English

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Research Area

Workflow Stages

Workflow Stages for Hematopoietic Stem and Progenitor Cell Research

Cell Sourcing and Isolation

Expansion and Differentiation

Analysis

Resources and Publications

Can EasySep™ be used for either positive or negative selection?

Yes. The EasySep™ kits use either a negative selection approach by targeting and removing unwanted cells or a positive selection approach targeting desired cells. Depletion kits are also available for the removal of cells with a specific undesired marker (e.g. GlyA).

How does the separation work?

Magnetic particles are crosslinked to cells using Tetrameric Antibody Complexes (TAC). When placed in the EasySep™ Magnet, labeled cells migrate to the wall of the tube. The unlabeled cells are then poured off into a separate fraction.

Which columns do I use?

The EasySep™ procedure is column-free. That's right - no columns!

How can I analyze the purity of my enriched sample?

The Product Information Sheet provided with each EasySep™ kit contains detailed staining information.

Can EasySep™ separations be automated?

Yes. RoboSep™, the fully automated cell separator, automates all EasySep™ labeling and cell separation steps.

Can EasySep™ be used to isolate rare cells?

Yes. We recommend a cell concentration of 2x10⁸ cells/mL and a minimum working volume of 100 µL. Samples containing 2x10⁷ cells or fewer should be suspended in 100 µL of buffer.

Are the EasySep™ magnetic particles FACS-compatible?

Yes, the EasySep™ particles are flow cytometry-compatible, as they are very uniform in size and about 5000X smaller than other commercially available magnetic beads used with column-free systems.

Can the EasySep™ magnetic particles be removed after enrichment?

No, but due to the small size of these particles, they will not interfere with downstream applications.

Can I alter the separation time in the magnet?

Yes; however, this may impact the kit's performance. The provided EasySep™ protocols have already been optimized to balance purity, recovery and time spent on the isolation.

For positive selection, can I perform more than 3 separations to increase purity?

Yes, the purity of targeted cells will increase with additional rounds of separations; however, cell recovery will decrease.

How does the binding of the EasySep™ magnetic particle affect the cells? is the function of positively selected cells altered by the bound particles?

Hundreds of publications have used cells selected with EasySep™ positive selection kits for functional studies. Our in-house experiments also confirm that selected cells are not functionally altered by the EasySep™ magnetic particles.

If particle binding is a key concern, we offer two options for negative selection. The EasySep™ negative selection kits can isolate untouched cells with comparable purities, while RosetteSep™ can isolate untouched cells directly from whole blood without using particles or magnets.

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions. K. Seiler et al. Cell death & disease 2022 may

Abstract

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.

View publication

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS-CoV-2 infection. R. M. van der Sluis et al. The EMBO journal 2022 may

Abstract

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID-19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFN? and of systemic inflammatory cytokines CXCL10 and IL-6. Using an in vitro stem cell-based human pDC model, we further demonstrate that pDCs, while not supporting SARS-CoV-2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFN? and IFN?1) and inflammatory (IL-6, IL-8, CXCL10) cytokines that protect epithelial cells from de novo SARS-CoV-2 infection. Via targeted deletion of virus-recognition innate immune pathways, we identify TLR7-MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll-like receptor (TLR)2 is responsible for the inflammatory IL-6 response. We further show that SARS-CoV-2 engages the receptor neuropilin-1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL-6 response, suggesting neuropilin-1 as potential therapeutic target for stimulation of TLR7-mediated antiviral protection.

View publication

Proton export alkalinizes intracellular pH and reprograms carbon metabolism to drive normal and malignant cell growth. C. H. Man et al. Blood 2022 jan

Abstract

Proton export is often considered a detoxifying process in animal cells, with monocarboxylate symporters coexporting excessive lactate and protons during glycolysis or the Warburg effect. We report a novel mechanism by which lactate/H+ export is sufficient to induce cell growth. Increased intracellular pH selectively activates catalysis by key metabolic gatekeeper enzymes HK1/PKM2/G6PDH, thereby enhancing glycolytic and pentose phosphate pathway carbon flux. The result is increased nucleotide levels, NADPH/NADP+ ratio, and cell proliferation. Simply increasing the lactate/proton symporter monocarboxylate transporter 4 (MCT4) or the sodium-proton antiporter NHE1 was sufficient to increase intracellular pH and give normal hematopoietic cells a significant competitive growth advantage in vivo. This process does not require additional cytokine triggers and is exploited in malignancy, where leukemogenic mutations epigenetically increase MCT4. Inhibiting MCT4 decreased intracellular pH and carbon flux and eliminated acute myeloid leukemia-initiating cells in mice without cytotoxic chemotherapy. Intracellular alkalization is a primitive mechanism by which proton partitioning can directly reprogram carbon metabolism for cell growth.

View publication

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EasySep™人脐带血CD34阳性选择试剂盒II

产品号 #（选择产品）

产品号 #17896_C

产品优势

产品组分包括

概述

Data Figures

Protocols and Documentation

Applications

Resources and Publications

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Scientists Helping Scientists™

Species	Human
Magnet Compatibility	• EasySep™ Magnet (Catalog #18000) • “The Big Easy” EasySep™ Magnet (Catalog #18001) • EasyEights™ EasySep™ Magnet (Catalog #18103) • RoboSep™-S (Catalog #21000)
Sample Source	Cord Blood
Selection Method	Positive

EasySep™人脐带血CD34阳性选择试剂盒II

产品号 #（选择产品）

产品号 #17896_C

产品优势

产品组分包括

概述

Data Figures

Protocols and Documentation

Applications

Resources and Publications

Educational Materials (9)

Frequently Asked Questions

Can EasySep™ be used for either positive or negative selection?

How does the separation work?

Which columns do I use?

How can I analyze the purity of my enriched sample?

Can EasySep™ separations be automated?

Can EasySep™ be used to isolate rare cells?

Are the EasySep™ magnetic particles FACS-compatible?

Can the EasySep™ magnetic particles be removed after enrichment?

Can I alter the separation time in the magnet?

For positive selection, can I perform more than 3 separations to increase purity?

How does the binding of the EasySep™ magnetic particle affect the cells? is the function of positively selected cells altered by the bound particles?

Publications (7)

Abstract

Abstract

Abstract

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