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Figure 1. Typical EasySep™ Human pDC Enrichment Profile
Starting with 0.2 - 0.9% pDC in PBMC, the pDC content of the enriched fraction typically ranges from 87 - 97% purity based on the pDC phenotype of Lineage (CD3, CD14, CD16, CD19, CD20, CD34, CD56) negative, HLA-DR positive, and CD304 (BDCA-4) positive.
Figure 2. FACS Purity Data from pDC Kit User
FACS enrichment plots from Dr. Stuart R. McGregor Dallas of Princeton University. Prior to enrichment, the percentage of pDCs in total PBMC is approximately 0.1% (upper row), however following enrichment, a population of pDCs in excess of 97% pure can be obtained (lower row). pDCs identified using surface marker staining for CD303 and HLA-DR. Data originally posted as part of a product review in Biocompare.
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.
Metabolic deficiencies underlie reduced plasmacytoid dendritic cell IFN-I production following viral infection
Nature Communications 2025 Feb
Abstract
Type I Interferons (IFN-I) are central to host protection against viral infections, with plasmacytoid dendritic cells (pDC) being the most significant source, yet pDCs lose their IFN-I production capacity following an initial burst of IFN-I, resulting in susceptibility to secondary infections. The underlying mechanisms of these dynamics are not well understood. Here we find that viral infection reduces the capacity of pDCs to engage both oxidative and glycolytic metabolism. Mechanistically, we identify lactate dehydrogenase B (LDHB) as a positive regulator of pDC IFN-I production in mice and humans; meanwhile, LDHB deficiency is associated with suppressed IFN-I production, pDC metabolic capacity, and viral control following infection. In addition, preservation of LDHB expression is sufficient to partially retain the function of otherwise exhausted pDCs, both in vitro and in vivo. Furthermore, restoring LDHB in vivo in pDCs from infected mice increases IFNAR-dependent, infection-associated pathology. Our work thus identifies a mechanism for balancing immunity and pathology during viral infections, while also providing insight into the highly preserved infection-driven pDC inhibition. Plasmacytoid dendritic cells (pDC) are the major IFN-I-producing cells, but this production returns to baseline soon after viral infection. Here the authors show that this decrease in IFN-I production and related pDC functions may be attributed to suppressed oxidative and glycolytic metabolism of pDCs, with lactate dehydrogenase B identified as a regulator.
The histamine analogue clobenpropit modulates IRF7 phosphorylation and interferon production by targeting CXCR4 in systemic lupus erythematosus models
Frontiers in Immunology 2024 Dec
Abstract
IntroductionSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by an overactive immune response, particularly involving excessive production of type I interferons. This overproduction is driven by the phosphorylation of IRF7, a crucial factor in interferon gene activation. Current treatments for SLE are often not very effective and can have serious side effects.MethodsOur study introduces clobenpropit, a histamine analogue, as a potential new therapy targeting the CXCR4 receptor to reduce IRF7 phosphorylation and subsequent interferon production. We employed various laboratory techniques to investigate how clobenpropit interacts with CXCR4 and its effects on immune cells from healthy individuals and SLE patients.ResultsClobenpropit binds effectively to CXCR4, significantly inhibiting IRF7 phosphorylation and reducing interferon production. Additionally, clobenpropit lowered levels of pro-inflammatory cytokines in a mouse model of lupus, demonstrating efficacy comparable to the standard treatment, prednisolone.DiscussionThese results suggest that clobenpropit could be a promising new treatment for SLE, offering a targeted approach with potential advantages over current therapies.
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.
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