EasySep™ Direct Human B Cell Isolation Kit

Immunomagnetic negative selection from whole blood kit

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EasySep™ Direct Human B Cell Isolation Kit

Immunomagnetic negative selection from whole blood kit

From: 567 USD
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Immunomagnetic negative selection from whole blood kit
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Product Advantages


  • > 99.9% RBC depletion without the need for density gradient centrifugation, sedimentation, or lysis

  • Up to 95% purity of isolated cells

  • Fast, easy-to-use and column-free

  • Isolated cells are untouched

What's Included

  • EasySep™ Direct Human B Cell Isolation Kit (Catalog #19674)
    • EasySep™ Direct Human B Cell Isolation Cocktail, 2 x 2.5 mL
    • EasySep™ Direct RapidSpheres™, 4 x 2.5 mL
  • RoboSep™ Human B Cell Isolation Kit (Catalog #19674RF)
    • EasySep™ Direct Human B Cell Isolation Cocktail, 2 x 2.5 mL
    • EasySep™ Direct RapidSpheres™, 4 x 2.5 mL
    • RoboSep™ Buffer (Catalog #20104)
    • RoboSep™ Filter Tips (Catalog #20125) x 2
Products for Your Protocol
To see all required products for your protocol, please consult the Protocols and Documentation.

Overview

Isolate highly purified B cells directly from human whole blood without the need for density gradient centrifugation, RBC sedimentation or lysis. This platform provides fast and fully immunomagnetic negative selection.

This cell isolation kit targets non-B cells for removal with EasySep™ Direct RapidSpheres™ and antibodies recognizing specific surface antigens. Unwanted cells are separated from desired cells using an EasySep™ magnet, and untouched target cells are then pipetted or poured into a new tube.

​Isolated cells can be used directly in downstream applications such as flow cytometry, culture or DNA/RNA extraction.
Magnet Compatibility
• EasySep™ Magnet (Catalog #18000)
• “The Big Easy” EasySep™ Magnet (Catalog #18001)
• Easy 50 EasySep™ Magnet (Catalog #18002)
• EasyEights™ EasySep™ Magnet (Catalog #18103)
• RoboSep™-S (Catalog #21000)
Subtype
Cell Isolation Kits
Cell Type
B Cells
Species
Human
Sample Source
Whole Blood
Selection Method
Negative
Application
Cell Isolation
Brand
EasySep, RoboSep
Area of Interest
Immunology

Data Figures

Typical EasySep™ Direct Human B Cell Isolation Profile

Figure 1. Typical EasySep™ Direct Human B Cell Isolation Profile

Starting with human whole blood from normal healthy donors, the typical B cell (CD3-CD19+) content of the non-lysed final isolated fraction is 95.3 ± 2.7% (gated on CD45) or 88.5 ± 11.5% (not gated on CD45). In the above example, the B cell (CD3-CD19+) content of the lysed whole blood start sample and the non-lysed final isolated fraction is 6.2% and 95.9% (gated on CD45), respectively, or 6.2% and 95.8% (not gated on CD45), respectively. The starting frequency of B cells in the non-lysed whole blood start sample above is 0.011% (data not shown).

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
Catalog #
19674
Lot #
All
Language
English
Catalog #
19674RF
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Catalog #
19674
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Catalog #
19674
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Catalog #
19674RF
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Catalog #
19674RF
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Catalog #
19674RF
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.

Resources and Publications

Frequently Asked Questions

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.

Publications (3)

A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells. Xiao X et al. mAbs 2016 JUL

Abstract

Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool.
FcγRIIB-Independent Mechanisms Controlling Membrane Localization of the Inhibitory Phosphatase SHIP in Human B Cells. Pauls SD et al. Journal of immunology (Baltimore, Md. : 1950) 2016 JUL

Abstract

SHIP is an important regulator of immune cell signaling that functions to dephosphorylate the phosphoinositide phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane and mediate protein-protein interactions. One established paradigm for SHIP activation involves its recruitment to the phospho-ITIM motif of the inhibitory receptor FcγRIIB. Although SHIP is essential for the inhibitory function of FcγRIIB, it also has critical modulating functions in signaling initiated from activating immunoreceptors such as B cell Ag receptor. In this study, we found that SHIP is indistinguishably recruited to the plasma membrane after BCR stimulation with or without FcγRIIB coligation in human cell lines and primary cells. Interestingly, fluorescence recovery after photobleaching analysis reveals differential mobility of SHIP-enhanced GFP depending on the mode of stimulation, suggesting that although BCR and FcγRIIB can both recruit SHIP, this occurs via distinct molecular complexes. Mutagenesis of a SHIP-enhanced GFP fusion protein reveals that the SHIP-Src homology 2 domain is essential in both cases whereas the C terminus is required for recruitment via BCR stimulation, but is less important with FcγRIIB coligation. Experiments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localization of SHIP, whereas neither PI3K or Src kinase activity is essential. BCR-induced association of SHIP with binding partner Shc1 is dependent on Syk, as is tyrosine phosphorylation of both partners. Our results indicate that FcγRIIB is not uniquely able to promote membrane recruitment of SHIP, but rather modulates its function via formation of distinct signaling complexes. Membrane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling.
Downregulation of MicroRNA-152 contributes to high expression of DKK1 in multiple myeloma. Y. Xu et al. RNA biology 2015

Abstract

Multiple myeloma (MM) induced bone lesion is one of the most crippling characteristics, and the MM secreted Dickkopf-1 (DKK1) has been reported to play important role in this pathologic process. However, the underlying regulation mechanisms involved in DKK1 expression are still unclear. In this study, we validated the expression patterns of microRNA (miR) 15a, 34a, 152, and 223 in MM cells and identified that miR-152 was significantly downregulated in the MM group compared with the non-MM group, and that miR-152 level was negatively correlated with the expression of DKK1 in the MM cells. Mechanistic studies showed that manipulating miR-152 artificially in MM cells led to changes in DKK-1 expression, and miR-152 blocked DKK1 transcriptional activity by binding to the 3'UTR of DKK1 mRNA. Importantly, we revealed that MM cells stably expressing miR-152 improved the chemotherapy sensitivity, and counteracted the bone disruption in an intrabone-MM mouse model. Our study contributes better understanding of the regulation mechanism of DKK-1 in MM, and opens up the potential for developing newer therapeutic strategies in the MM treatment.
New look, same high quality and support! You may notice that your instrument or reagent packaging looks slightly different from images displayed on the website, or from previous orders. We are updating our look but rest assured, the products themselves and how you should use them have not changed. Learn more