RosetteSep™ HLA Lymphoid Cell Enrichment Kit

Immunodensity negative selection cocktail

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RosetteSep™ HLA Lymphoid Cell Enrichment Kit

Immunodensity isolation of untouched CD3+ lymphocytes for lineage-specific chimerism analysis

For 20 Tests (10 mL of whole blood per test)
Catalog #15271HLA
915 USD

Required Products

Overview

The RosetteSep™ HLA Lymphoid Cell Enrichment Cocktail is designed to enrich lymphoid (CD3+) cells from whole blood by negative selection for lineage-specific chimerism analysis. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing CD16, CD19, CD20, CD36, CD56 and glycophorin A on red blood cells (RBCs). When centrifuged over a buoyant density medium such as RosetteSep™ DM-L (Catalog #15705) the unwanted cells pellet along with the RBCs. The purified lymphoid cells are present as a highly enriched population at the interface between the plasma and the buoyant density medium. The desired cells are immediately ready for chimerism analysis. This kit carries the CE mark and is available as a Class I in vitro diagnostic device in the European Union and Australia. To learn more about the regulatory status of this product in your specific region, contact info@stemcell.com.
Advantages:
• Fast and easy-to-use
• Requires no special equipment or training
• Isolated cells are untouched
Components:
  • RosetteSep™ HLA Lymphoid Cell Enrichment Kit (Catalog #15271HLA)
    • RosetteSep™ HLA Lymphoid Cell Enrichment Cocktail, 5 x 2 mL
    • RosetteSep™ DM-L, (Catalog #15705) x 2
Subtype:
Cell Isolation Kits
Cell Type:
T Cells
Species:
Human
Sample Source:
Buffy Coat; Whole Blood
Selection Method:
Negative
Application:
Cell Isolation; In Vitro Diagnostic
Brand:
RosetteSep
Area of Interest:
Chimerism; HLA; Immunology

Scientific Resources

Educational Materials

(4)

Frequently Asked Questions

What is RosetteSep™?

RosetteSep™ is a rapid cell separation procedure for the isolation of purified cells directly from whole blood, without columns or magnets.

How does RosetteSep™ work?

The antibody cocktail crosslinks unwanted cells to red blood cells (RBCs), forming rosettes. The unwanted cells then pellet with the free RBCs when centrifuged over a density centrifugation medium (e.g. Ficoll-Paque™ PLUS, Lymphoprep™).

What factors affect cell recovery?

The temperature of the reagents can affect cell recovery. All reagents should be at room temperature (sample, density centrifugation medium, PBS, centrifuge) before performing the isolations. Layering can also affect recovery so be sure to carefully layer the sample to avoid mixing with the density centrifugation medium as much as possible. Be sure to collect the entire enriched culture without disturbing the RBC pellet. A small amount of density centrifugation medium can be collected without worry.

Which cell samples can RosetteSep™ be used with?

RosetteSep™ can be used with leukapheresis samples, bone marrow or buffy coat, as long as: the concentration of cells does not exceed 5 x 107 per mL (can dilute if necessary); and there are at least 100 RBCs for every nucleated cell (RBCs can be added if necessary).

Can RosetteSep™ be used with previously frozen or cultured cells?

Yes. Cells should be re-suspended at 2 - 5 x 107 cells / mL in PBS + 2% FBS. Fresh whole blood should be added at 250 µL per mL of sample, as a source of red cells.

Can RosetteSep™ be used to enrich progenitors from cord blood?

Yes. Sometimes cord blood contains immature nucleated red cells that have a lower density than mature RBCs. These immature red cells do not pellet over Ficoll™, which can lead to a higher RBC contamination than peripheral blood separations.

Does RosetteSep™ work with mouse cells?

No, but we have developed EasySep™, a magnetic-based cell isolation system which works with mouse and other non-human species.

Which anticoagulant should be used with RosetteSep™?

Peripheral blood should be collected in heparinized Vacutainers. Cord blood should be collected in ACD.

Should the anticoagulant be washed off before using RosetteSep™?

No, the antibody cocktail can be added directly to the sample.
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Product 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.

Data and Publications

Data

FACS Histogram Results with RosetteSep™ HLA Lymphoid Cell Enrichment Kit

Figure 1. FACS Histogram Results with RosetteSep™ HLA Lymphoid Cell Enrichment Kit

Publications

(2)
Journal of immunology (Baltimore, Md. : 1950) 2005 FEB

Dendritic cells induce MUC1 expression and polarization on human T cells by an IL-7-dependent mechanism.

Vasir B et al.

Abstract

The MUC1 transmembrane mucin is expressed on the surface of activated human T cells; however, the physiologic signals responsible for the regulation of MUC1 in T cells are not known. The present studies demonstrate that IL-7, but not IL-2 or IL-4, markedly induces MUC1 expression on CD3+ T cells. MUC1 was also up-regulated by IL-15, but to a lesser extent than that found with IL-7. The results show that IL-7 up-regulates MUC1 on CD4+, CD8+, CD25+, CD69+, naive CD45RA+, and memory CD45RO+ T cells. In concert with induction of MUC1 expression by IL-7, activated dendritic cells (DC) that produce IL-7 up-regulate MUC1 on allogeneic CD3+ T cells. DC also induce MUC1 expression on autologous CD3+ T cells in the presence of recall Ag. Moreover, DC-induced MUC1 expression on T cells is blocked by a neutralizing anti-IL-7 Ab. The results also demonstrate that DC induce polarization of MUC1 on T cells at sites opposing the DC-T cell synapse. These findings indicate that DC-mediated activation of Ag-specific T cells is associated with induction and polarization of MUC1 expression by an IL-7-dependent mechanism.
Blood 2003 NOV

Rapid induction of complete donor chimerism by the use of a reduced-intensity conditioning regimen composed of fludarabine and melphalan in allogeneic stem cell transplantation for metastatic solid tumors.

Ueno NT et al.

Abstract

We evaluated the feasibility and efficacy of a reduced-intensity conditioning (RIC) regimen of fludarabine and melphalan to achieve rapid complete donor chimerism after allogeneic stem cell transplantation (SCT) in patients with metastatic solid tumors. Between January 1999 and January 2003, 8 patients with metastatic breast cancer (BC) and 15 with metastatic renal cell carcinoma (RCC) underwent allogeneic SCT after an RIC regimen of 5 days of fludarabine and 2 days of melphalan. Filgrastim-mobilized stem cells from HLA-identical related or unrelated donors were infused. Prophylaxis for graft-versus-host disease (GVHD) consisted of tacrolimus and methotrexate. All 22 evaluable patients had 100% donor chimerism at day 30 and at all measurement times thereafter. One patient died 19 days after SCT. Nine patients (39%) had grades II to IV acute GVHD and 10 patients (43%) had chronic GVHD. Five patients (22%) died of nonrelapse treatment-related complications. Treatment-related disease response was seen in 10 patients (45%), with 3 complete responses, 2 partial responses, and 5 minor responses. Fludarabine-melphalan is a feasible and effective RIC regimen for allogeneic SCT in metastatic BC and RCC. It induces rapid complete donor chimerism without the need for donor lymphocyte infusion. Tumor regression associated with GVHD is consistent with graft-versus-tumor effect.
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