Figure 1. FACS Histogram Results Using RosetteSep™ Human CD4+ T Cell Enrichment Cocktail
Starting with fresh human whole blood, the CD4+ (CD3+CD4+) T cell content of the enriched fraction is typically 94.7 ± 2.4% (mean ± SD). In the above example, the purities of the start and final enriched fractions are 15.1% and 95.0%, respectively.
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.
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.
Vpu modulates DNA repair to suppress innate sensing and hyper-integration of HIV-1. M. Volcic et al. Nature microbiology 2020 oct
Abstract
To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs.
Potent inhibition of HIV replication in primary human cells by novel synthetic polyketides inspired by Aureothin A. Herrmann et al. Scientific Reports 2020 jan
Abstract
Overcoming the global health threat of HIV infection requires continuous pipelines of novel drug candidates. We identified the $\gamma$-pyrone polyketides Aureothin/Neoaureothin as potent hits by anti-HIV screening of an extensive natural compound collection. Total synthesis of a structurally diverse group of Aureothin-derivatives successfully identified a lead compound ({\#}7) superior to Aureothin that combines strong anti-HIV activity (IC90{\textless}45 nM), photostability and improved cell safety. Compound {\#}7 inhibited de novo virus production from integrated proviruses by blocking the accumulation of HIV RNAs that encode the structural components of virions and include viral genomic RNAs. Thus, the mode-of-action displayed by compound {\#}7 is different from those of all current clinical drugs. Proteomic analysis indicated that compound {\#}7 does not affect global protein expression in primary blood cells and may modulate cellular pathways linked to HIV infection. Compound {\#}7 inhibited multiple HIV genotypes, including HIV-type 1 and 2 and synergistically inhibited HIV in combination with clinical reverse transcriptase and integrase inhibitors. We conclude that compound {\#}7 represents a promising new class of HIV inhibitors that will facilitate the identification of new virus-host interactions exploitable for antiviral attack and holds promise for further drug development.
Nef-Mediated CD3-TCR Downmodulation Dampens Acute Inflammation and Promotes SIV Immune Evasion. S. Joas et al. Cell reports 2020 feb
Abstract
The inability of Nef to downmodulate the CD3-T cell receptor (TCR) complex distinguishes HIV-1 from other primate lentiviruses and may contribute to its high virulence. However, the role of this Nef function in virus-mediated immune activation and pathogenicity remains speculative. Here, we selectively disrupted this Nef activity in SIVmac239 and analyzed the consequences for the virological, immunological, and clinical outcome of infection in rhesus macaques. The inability to downmodulate CD3-TCR does not impair viral replication during acute infection but is associated with increased immune activation and antiviral gene expression. Subsequent early reversion in three of six animals suggests strong selective pressure for this Nef function and is associated with high viral loads and progression to simian AIDS. In the absence of reversions, however, viral replication and the clinical course of infection are attenuated. Thus, Nef-mediated downmodulation of CD3 dampens the inflammatory response to simian immunodeficiency virus (SIV) infection and seems critical for efficient viral immune evasion.
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EasySep™小鼠TIL（CD45）正选试剂盒
