Figure 1. FACS Histogram Results Using EasySep™ Human Monocyte Enrichment Kit Without CD16 Depletion
Starting with freshly prepared PBMCs, the CD14+ cell content of the enriched fraction is 82.7 ± 6.7% (mean ± SD using the purple EasySep™ magnet). Slightly lower CD14+ cell purities may be obtained from samples that contain a large number of CD16+ cells. In the above example, the start and final enriched fractions are 21.2% and 83.3%, respectively.
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.
Type I interferons increase expression of endogenous retrovirus K102 and envelope protein in myeloid cells from patients with autoimmune disease
E. Le et al.
Mobile DNA 2025 Sep
Abstract
BackgroundAutoantibodies against envelope (Env) protein encoded by human endogenous retrovirus group K (HERV-K) are prevalent in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), but it remains unclear which proviruses are responsible for this autoantigen. It also remains poorly understood how the transcription of HERV-K loci is regulated in cells that can produce Env.ResultsWe aligned our neutrophil RNA sequencing data to the new telomere-to-telomere reference genome and found uniquely mapping transcripts from HERV-K101, K102, K104, K108, K109, K117 and ERVK5, of which only K102, K108, and K109 encode an intact Env. Expression of K102 and K108 were higher in SLE than in healthy donors or RA (padj < 0.05). Transcripts from these proviruses increased in response to interferon-α in monocytes and neutrophils from RA patients and healthy donors, but not in SLE, presumably because they have chronically elevated type I interferons in vivo. Indeed, HERV-K expression was significantly higher in SLE patients with high type I interferon gene signature. Tumor necrosis factor-α and other cytokines and TLR ligands also induced HERV-K102 and K108 transcripts. Interferon-α also increased detectable Env protein in monocytes, macrophages, and neutrophils from RA patients. Among the genes for epigenetic silencers of HERV-K, only TRIM28 was significantly decreased in SLE patients with high interferons (padj = 0.00024).ConclusionsOur data establish a role for interferons in maintaining increased HERV-K expression in SLE and suggest that interferons or other cytokines can upregulate HERV-K to similar levels in RA. A transient increase may also accompany normal immune responses, suggesting that endogenous retroviruses may have been co-opted for efficient immune responses.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13100-025-00371-y. Key points
Expression of HERV-K provirus is elevated in neutrophils from IFN-positive SLETNFα, IFN, and other cytokines induce similar HERV-K expression also in RAHealthy donor myeloid cells respond only transiently with HERV-K transcription
Supplementary InformationThe online version contains supplementary material available at 10.1186/s13100-025-00371-y.
Lifting the curse from high-dimensional data: automated projection pursuit clustering for a variety of biological data modalities
GigaScience 2025 May
Abstract
AbstractUnsupervised clustering is a powerful machine-learning technique widely used to analyze high-dimensional biological data. It plays a crucial role in uncovering patterns, structures, and inherent relationships within complex datasets without relying on predefined labels. In the context of biology, high-dimensional data may include transcriptomics, proteomics, and a variety of single-cell omics data. Most existing clustering algorithms operate directly in the high-dimensional space, and their performance may be negatively affected by the phenomenon known as the curse of dimensionality. Here, we show an alternative clustering approach that alleviates the curse by sequentially projecting high-dimensional data into a low-dimensional representation. We validated the effectiveness of our approach, named automated projection pursuit (APP), across various biological data modalities, including flow and mass cytometry data, scRNA-seq, multiplex imaging data, and T-cell receptor repertoire data. APP efficiently recapitulated experimentally validated cell-type definitions and revealed new biologically meaningful patterns.
Monocyte subsets in breast cancer patients under treatment with aromatase inhibitor and mucin-1 cancer vaccine
Journal of Translational Medicine 2024 Oct
Abstract
BackgroundMonocytes comprise subsets of classical, intermediate and non-classical monocytes with distinct anti- or pro-tumor effects in breast cancer (BC). They are modulated by estrogen, and can contribute to BC control by endocrine therapy in preclinical models.MethodsTo elucidate whether changes in monocyte subsets are associated with treatment and response, we investigated peripheral blood samples of 73 postmenopausal women with estrogen receptor (ER) positive BC, who received aromatase inhibitor therapy with or without the mucin-1 vaccine tecemotide in the ABCSG34 trial. Blood was retrieved at baseline, midterm and end of therapy, and was analyzed for the distribution and ER expression of monocyte subsets by flow cytometry.ResultsWhen 40 healthy, age-matched women were compared with BC patients before treatment start, ER levels of monocytes did not differ, yet patients presented with a higher frequency of classical and fewer non-classical monocytes. Endocrine therapy triggered a significant increase in ER levels in all monocyte subsets, without affecting subset distribution. Vaccination had no overall impact on subset frequency and ER expression. Yet, a shift from intermediate to classical monocytes during therapy correlated with changes in plasma cytokines and chemokines and was significantly associated with low residual cancer burden in vaccinated patients. Without tecemotide, baseline ER levels in classical monocytes were significantly higher in women with good response to endocrine therapy.ConclusionsThis study identified classical monocytes to be associated with ER positive BC and with patient response to neoadjuvant endocrine treatment and cancer vaccination.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-024-05659-w.
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