Isolate highly purified human CD4+ cells from fresh or previously frozen human peripheral blood mononuclear cells (PBMCs) or washed leukapheresis samples by immunomagnetic positive selection, with the EasySep™ Human CD4 Positive Selection Kit II. Widely used in published research for more than 20 years, EasySep™ combines the specificity of monoclonal antibodies with the simplicity of a column-free magnetic system.
In this EasySep™ positive selection procedure, desired cells are labeled with antibody complexes recognizing CD4 and magnetic particles. Labeled cells are separated using an EasySep™ magnet and by simply pouring or pipetting off the unwanted cells. The cells of interest remain in the tube. Following magnetic cell isolation in as little as 15 minutes, the desired CD4+ cells are ready for downstream applications such as flow cytometry, culture, or DNA/RNA extraction.
This product replaces the EasySep™ Human CD4 Positive Selection Kit (Catalog #18052) for even faster cell isolations.
For large-scale isolation of CD4+ cells from leukapheresis samples, see the large-format (1x10^10 cells) kit (Catalog #100-0693).
Learn more about how immunomagnetic EasySep™ technology works or how to fully automate immunomagnetic cell isolation with RoboSep™. Explore additional products optimized for your workflow, including culture media, supplements, antibodies, and more.
(C) Flow cytometry analysis of human PBMCs isolated with the EasySep™ Human CD4 Positive Selection Kit (Catalog #17852) and labeled with Anti-Human CD4 Antibody, Clone OKT4, Alexa Fluor® 488. Histograms show labeling of total PBMCs (Start) and isolated cells (Isolated). Labeling with Mouse IgG2b, kappa Isotype Control Antibody, Clone MPC-11, Alexa Fluor® 488 is shown in the bottom panel (solid line histogram).
Figure 3. Gene Expression Profiles of EasySep™-Isolated CD4+ T Cells Are Similar to PBMC Control
(A,B) tSNE plots were generated using data from (A) PBMC control or (B) cells isolated using the EasySep™ Human CD4+ T Cell Enrichment Kit (Catalog #19052). CD4+ T cell clusters are colored as indicated in the legend.
(C,D) 500 genes were selected from a previously published list of CD4+ T cell signature markers (Zhang et al., 2018). Expression heatmaps were generated for CD4+ cells from (C) PBMC control and (D) cells isolated using the EasySep™ Human CD4 Positive Selection Kit II (Catalog #17852), EasySep™ Human CD4+ T Cell Enrichment Kit (Catalog #19052), or the EasySep™ Release Human CD4 Positive Selection Kit (Catalog #17752). The average expression was calculated within each sample for three CD4+ T cell clusters identified by Seurat (naïve, central memory, and effector memory CD4 T+ cells).
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.
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.
Engagement of TRAIL triggers degranulation and IFN$\gamma$ production in human natural killer cells. J. H\ofle et al." EMBO reports 2022 aug
Abstract
NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus-infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV-1-infected cells. By combining an unbiased large-scale screening approach with a functional assay, we identify TRAIL to be associated with NK cell degranulation against HIV-1-infected target cells. Further investigating the underlying mechanisms, we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor-mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFN$\gamma$ production. Moreover, TRAIL-mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I, adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL-mediated cytotoxicity. Based on these findings, we propose that TRAIL not only contributes to the anti-HIV-1 activity of NK cells but also possesses a multifunctional role beyond receptor-mediated induction of apoptosis, acting as a regulator for the induction of different effector functions.
Targeting 3D chromosomal architecture at the RANK loci to suppress myeloma-driven osteoclastogenesis. K. Th\ummler et al." Oncoimmunology 2022
Abstract
Bone disease represents a major cause of morbidity and mortality in Multiple Myeloma (MM); primarily driven by osteoclasts whose differentiation is dependent on expression of RANKL by MM cells. Notably, costimulation by ITAM containing receptors (i.e., Fc$\gamma$R) can also play a crucial role in osteoclast differentiation. Modeling the pathology of the bone marrow microenvironment with an ex vivo culture system of primary human multiple myeloma cells, we herein demonstrate that Fc$\gamma$R-mediated signaling, via staphylococcal protein A (SpA) IgG immune-complexes, can act as a critical negative regulator of MM-driven osteoclast differentiation. Interrogation of the mode-of-action revealed that Fc$\gamma$R-mediated signaling causes epigenetic modulation of chromosomal 3D architecture at the RANK promoter; with altered spatial orientation of a proximal super enhancer. Combined this leads to substantial down-regulation of RANK at a transcript, protein, and functional level. These observations shed light on a novel mechanism regulating RANK expression and provide a rationale for targeting Fc$\gamma$R-signaling for the amelioration of osteolytic bone pathology in disease.
Competition between PAF1 and MLL1/COMPASS confers the opposing function of LEDGF/p75 in HIV latency and proviral reactivation. R. Gao et al. Science advances 2020 may
Abstract
Transcriptional status determines the HIV replicative state in infected patients. However, the transcriptional mechanisms for proviral replication control remain unclear. In this study, we show that, apart from its function in HIV integration, LEDGF/p75 differentially regulates HIV transcription in latency and proviral reactivation. During latency, LEDGF/p75 suppresses proviral transcription via promoter-proximal pausing of RNA polymerase II (Pol II) by recruiting PAF1 complex to the provirus. Following latency reversal, MLL1 complex competitively displaces PAF1 from the provirus through casein kinase II (CKII)-dependent association with LEDGF/p75. Depleting or pharmacologically inhibiting CKII prevents PAF1 dissociation and abrogates the recruitment of both MLL1 and Super Elongation Complex (SEC) to the provirus, thereby impairing transcriptional reactivation for latency reversal. These findings, therefore, provide a mechanistic understanding of how LEDGF/p75 coordinates its distinct regulatory functions at different stages of the post-integrated HIV life cycles. Targeting these mechanisms may have a therapeutic potential to eradicate HIV infection.
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