RosetteSep™ Human T Cell Enrichment Cocktail

Immunodensity negative selection cocktail

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Immunodensity negative selection cocktail
From: 174 USD

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Overview

The RosetteSep™ Human T Cell Enrichment Cocktail is designed to isolate T cells from whole blood by negative selection. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing non-T cells and glycophorin A on red blood cells (RBCs). When centrifuged over a buoyant density medium such as RosetteSep™ DM-L (Catalog #15705) or Lymphoprep™ (Catalog #07801), the unwanted cells pellet along with the RBCs. The purified T cells are present as a highly enriched population at the interface between the plasma and the buoyant density medium.
Advantages:
• Fast and easy-to-use
• Requires no special equipment or training
• Isolated cells are untouched
• Can be combined with SepMate™ for consistent, high-throughput sample processing
Components:
  • RosetteSep™ Human T Cell Enrichment Cocktail (Catalog #15021)
    • RosetteSep™ Human T Cell Enrichment Cocktail, 2 mL
  • RosetteSep™ Human T Cell Enrichment Cocktail (Catalog #15061)
    • RosetteSep™ Human T Cell Enrichment Cocktail, 5 x 2 mL
Subtype:
Cell Isolation Kits
Cell Type:
T Cells
Species:
Human
Sample Source:
Buffy Coat; Whole Blood
Selection Method:
Negative
Application:
Cell Isolation
Brand:
RosetteSep
Area of Interest:
Immunology

Scientific Resources

Educational Materials

(9)

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

Typical RosetteSep™ HLA T Cell Enrichment Profile

Figure 1. Typical RosetteSep™ T Cell Enrichment Profile

Starting with fresh whole blood the CD3+ cell content of the enriched fraction typically ranges from 90% - 97%. Red blood cells were removed by lysis prior to flow cytometry.

Publications

(46)
Scientific reports 2019 may

Differences in the molecular signatures of mucosal-associated invariant T cells and conventional T cells.

D. Park et al.

Abstract

Mucosal-associated invariant T (MAIT) cells exhibit different characteristics from those of TCRalpha7.2- conventional T cells. They play important roles in various inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease. MAIT cells express a single T cell receptor alpha chain, TCRalpha7.2 segment associated with Jalpha33 and CDR3 with fixed length, which recognizes bacteria-derived vitamin B metabolites. However, the characteristics of MAIT cells and TCRalpha7.2+ CD161- T cells have never been compared. Here, we performed RNA sequencing to compare the properties of MAIT cells, TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells. Genome-wide transcriptomes of MAIT cells, TCRalpha7.2- conventional T cells, and TCRalpha7.2+ CD161- T cells were compared and analyzed using causal network analysis. This is the first report comparing the transcriptomes of MAIT cells, TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells. We also identified the predominant signaling pathways of MAIT cells, which differed from those of TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells, through a gene set enrichment test and upstream regulator analysis and identified the genes responsible for the characteristic MAIT cell phenotypes. Our study advances the complete understanding of MAIT biology.
Nature biotechnology 2019 feb

Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.

B. P. Kleinstiver et al.

Abstract

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases1 has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)2. To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants3 to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.
Nature medicine 2018 OCT

Translational control of tumor immune escape via the eIF4F-STAT1-PD-L1 axis in melanoma.

M. Cerezo et al.

Abstract

Preventing the immune escape of tumor cells by blocking inhibitory checkpoints, such as the interaction between programmed death ligand-1 (PD-L1) and programmed death-1 (PD-1) receptor, is a powerful anticancer approach. However, many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker, but the complex mechanisms underlying its regulation are not completely understood. Here, we show that the eukaryotic translation initiation complex, eIF4F, which binds the 5' cap of mRNAs, regulates the surface expression of interferon-$\gamma$-induced PD-L1 on cancer cells by regulating translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor. eIF4F complex formation correlates with response to immunotherapy in human melanoma. Pharmacological inhibition of eIF4A, the RNA helicase component of eIF4F, elicits powerful antitumor immune-mediated effects via PD-L1 downregulation. Thus, eIF4A inhibitors, in development as anticancer drugs, may also act as cancer immunotherapies.
Journal of immunological methods 2017 MAR

T cell activation and proliferation following acute exercise in human subjects is altered by storage conditions and mitogen selection.

Siedlik JA et al.

Abstract

Recent work investigating exercise induced changes in immunocompetence suggests that some of the ambiguity in the literature is resultant from different cell isolation protocols and mitogen selection. To understand this effect, we compared post-exercise measures of T cell activation and proliferation using two different stimulation methods (costimulation through CD28 or stimulation with phytohaemagglutinin [PHA]). Further, we investigated whether exercise induced changes are maintained when T cell isolation from whole blood is delayed overnight in either a room temperature or chilled (4°C) environment. As expected, an increased proliferation response was observed post-exercise in T cells isolated from whole blood of previously trained individuals immediately after blood collection. Also, cells stimulated with PHA after resting overnight in whole blood were not adversely impacted by the storage conditions. In contrast, allowing cells to rest overnight in whole blood prior to stimulation through CD28, lessened the proliferation observed by cells following exercise rendering both the room temperature and chilled samples closer to the results seen in the control condition. Changes in early markers of activation (CD25), followed a similar pattern, with activation in PHA stimulated cells remaining fairly robust after overnight storage; whereas cell activation following stimulation through CD3+CD28 was disproportionately decreased by the influence of overnight storage. These findings indicate that decisions regarding cell stimulation methods need to be paired with the timeline for T cell isolation from whole blood. These considerations will be especially important for field based studies of immunocompetence where there is a delay in getting whole blood samples to a lab for processing as well as clinical applications where a failure to isolate T cells in a timely manner may result in loss of the response of interest.
PloS one 2017

Vitamin D receptor gene is epigenetically altered and transcriptionally up-regulated in multiple sclerosis.

Ayuso T et al.

Abstract

OBJECTIVE Vitamin D deficiency has been linked to increased risk of multiple sclerosis (MS) and poor outcome. However, the specific role that vitamin D plays in MS still remains unknown. In order to identify potential mechanisms underlying vitamin D effects in MS, we profiled epigenetic changes in vitamin D receptor (VDR) gene to identify genomic regulatory elements relevant to MS pathogenesis. METHODS Human T cells derived from whole blood by negative selection were isolated in a set of 23 relapsing-remitting MS (RRMS) patients and 12 controls matched by age and gender. DNA methylation levels were assessed by bisulfite cloning sequencing in two regulatory elements of VDR. mRNA levels were measured by RT-qPCR to assess changes in VDR expression between patients and controls. RESULTS An alternative VDR promoter placed at exon 1c showed increased DNA methylation levels in RRMS patients (median 30.08%, interquartile range 19.2%) compared to controls (18.75%, 9.5%), p-valuetextless0.05. Moreover, a 6.5-fold increase in VDR mRNA levels was found in RRMS patients compared to controls (p-valuetextless0.001). CONCLUSIONS An alternative promoter of the VDR gene shows altered DNA methylation levels in patients with multiple sclerosis, and it is associated with VDR mRNA upregulation. This locus may represent a candidate regulatory element in the genome relevant to MS pathogenesis.
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.