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What Our Scientist Says
Isolating immune cells can be time consuming, but it doesn't have to be. That's why we developed a wide variety of EasySep™ kits, including this 12.5-minute monocyte isolation kit.
Figure 1. Typical EasySep™ Human Monocyte Isolation Kit (Catalog #19359)
Starting with PBMCs prepared from human peripheral blood, the monocyte cell content (CD14+CD16-) of the isolated fraction obtained without (middle plots) or with (bottom plots) EasySep™ Human Platelet Removal Cocktail is typically 89.7 ± 3.4% and 87.3 ± 4.5%, respectively (gated on CD45; mean ± SD for the purple EasySep™ Magnet). In the above example, the purities of the start and final isolated fractions obtained without (middle plot) or with (bottom plots) the EasySep™ Human Platelet Removal Cocktail are 20.0%, 88.2%, and 84.8%, respectively (gated on CD45) and 18.0%, 55.7%, and 71.7% (not gated on CD45).
Figure 2. Cryopreserved Monocytes Differentiate into Dendritic Cells and Secrete IL-12 (p70) and IL-23 Upon Activation
Monocytes freshly isolated from a leukopak (Catalog #70500) using EasySep™ Human Monocyte Isolation Kit (Catalog #19359) or cryopreserved monocytes (Catalog #70034) were cultured for 6 days in RPMI 1640 Medium (Catalog #36750) with 10% FBS, 0.1 mM MEM Non-Essential Amino Acid Solution (100X, Catalog #07600), 2 mM L-Glutamine (Catalog #07100), 1 mM Sodium Pyruvate (Catalog #07000), and 50 µM β-mercaptoethanol. Human Recombinant IL-4 (Catalog #78045) and Human Recombinant GM-CSF (Catalog #78015) were added on Days 1, 3, and 6 to differentiate monocytes into DCs. Cells were either left unstimulated (control) or stimulated with LPS and Human Recombinant IFN-γ (Catalog #78020) (activated). Activation led to secretion of (A) IL-12 (p70) and (B) IL-23, which were not detectable in unstimulated controls as measured using the Human IL-12 (p70) ELISA Kit (Catalog #02014) and the Human IL-23 ELISA Kit (Catalog #02016),respectively. *Cytokine concentration of control in culture was lower than the limit of detection.
Figure 3. Mature DCs Generated with ImmunoCult™-ACF Dendritic Cell Medium with Supplements Show Desired Phenotype
Monocytes isolated using EasySep™ Human Monocyte Isolation Kit (Catalog #19359) were cultured and differentiated into mature dendritic cells (DCs) as described in the PIS for ImmunoCult™-ACF Dendritic Cell Culture Kit (Catalog #10985). (A) The percentage of CD14 and CD83 expression in cells at Day 7 (mature DCs) was determined by flow cytometry. At Day 7, a total of 93 ± 5% of the cells expressed the mature DC marker CD83 and only 1 ± 1% of cells still expressed the monocyte marker CD14 (mean ± SD, n = 39). The yield of mature DCs was determined by the count of total viable cells at Day 7 relative to the count of viable monocytes used for initial culture at Day 0. At Day 7, the yield of viable mature DCs corresponded to 45 ± 25% (mean ± SD, n=39). (B) Immature DCs were cultured as described in Figure 1. At Day 5, cells were cultured with maturation supplement for 2 days (mature DCs) or without maturation supplement (immature DCs). Supernatant was collected at Day 7 and IL-12p70 levels were determined by ELISA. Concentrations of IL-12p70 in supernatant of mature and immature DCs were 361 ± 81 and 5 ± 2 pg/mL, respectively (mean ± SEM, n = 27).
Figure 4. Refrigeration of Leukopaks Preserves Monocyte-to-Macrophage Differentiation Efficiency for Up to 5 Days
Using EasySep™ Human Monocyte Isolation Kit (Catalog #19359), monocytes were isolated from 1 leukopak fraction (Catalog #70500) of each storage condition daily for 5 days, and 1 x 10⁶ isolated cells were cultured in ImmunoCult™-SF Macrophage Medium (Catalog #10961) supplemented with 50 ng/mL Human Recombinant M-CSF (Catalog #78057) for a further 6 days. (A) Representative flow cytometry plot from leukopaks stored 1 day at fridge temperature (FT), showing maintenance of CD14 and upregulation of CD68 expression over the 5 day differentiation to M0 macrophages. (B) While monocytes isolated from FT-stored leukopaks efficiently differentiated into M0 macrophages, those stored at room temperature (RT) for over 3 days failed to differentiate, as shown by low percentages of CD14+CD68+ cells. Moreover, monocytes harvested from Day 5 leukopaks stored at RT failed to thrive in the 6-day culture, resulting in very few viable cells recovered (not shown). All data points represent average ± standard deviation values from leukopak fractions of n = 3 unique donors.
Figure 5. Refrigeration of Leukopaks Preserves Monocyte-to-Dendritic Cell Differentiation Ability for Up to 5 Days
Using EasySep™ Human Monocyte Isolation Kit (Catalog #19359), monocytes were isolated from 1 leukopak fraction (Catalog #70500) of each storage condition daily for 5 days, and 1 x 10⁶ isolated cells were cultured in ImmunoCult™-ACF Dendritic Cell (DC) Medium (Catalog #10986) supplemented with ImmunoCult™-ACF Dendritic Cell Differentiation Supplement (Catalog #10988). (A) Representative flow cytometry plot from leukopaks stored 1 day at fridge temperature (FT), showing efficient downregulation of CD14 and upregulation of CD11c over 6 days of culture. (B) While monocytes isolated from FT-stored leukopaks efficiently differentiated into DCs, those stored at room temperature (RT) for over 3 days show reduced DC differentiation ability and CD14-CD11c+ cell output. Moreover, monocytes harvested from day 5 RT-stored leukopaks failed to thrive in the 5-day culture, resulting in very few viable cells recovered (not shown). All data points represent average ± standard deviation values from leukopak fractions of n = 3 unique donors.
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.
Chemoproteomic development of SLC15A4 inhibitors with anti-inflammatory activity. T.-Y. Chiu et al. Nature chemical biology 2024 aug
Abstract
SLC15A4 is an endolysosome-resident transporter linked with autoinflammation and autoimmunity. Specifically, SLC15A4 is critical for Toll-like receptors (TLRs) 7-9 as well as nucleotide-binding oligomerization domain-containing protein (NOD) signaling in several immune cell subsets. Notably, SLC15A4 is essential for the development of systemic lupus erythematosus in murine models and is associated with autoimmune conditions in humans. Despite its therapeutic potential, the availability of quality chemical probes targeting SLC15A4 functions is limited. In this study, we used an integrated chemical proteomics approach to develop a suite of chemical tools, including first-in-class functional inhibitors, for SLC15A4. We demonstrate that these inhibitors suppress SLC15A4-mediated endolysosomal TLR and NOD functions in a variety of human and mouse immune cells; we provide evidence of their ability to suppress inflammation in vivo and in clinical settings; and we provide insights into their mechanism of action. Our findings establish SLC15A4 as a druggable target for the treatment of autoimmune and autoinflammatory conditions.
Human alveolar macrophages display marked hypo-responsiveness to IFN-$\gamma$ in both proteomic and gene expression analysis. B. A. Thiel et al. PloS one 2024
Abstract
Alveolar macrophages (AM) perform a primary defense mechanism in the lung through phagocytosis of inhaled particles and microorganisms. AM are known to be relatively immunosuppressive consistent with the aim to limit alveolar inflammation and maintain effective gas exchange in the face of these constant challenges. How AM respond to T cell derived cytokine signals, which are critical to the defense against inhaled pathogens, is less well understood. For example, successful containment of Mycobacterium tuberculosis (Mtb) in lung macrophages is highly dependent on IFN-$\gamma$ secreted by Th-1 lymphocytes, however, the proteomic IFN-$\gamma$ response profile in AM remains mostly unknown. In this study, we measured IFN-$\gamma$ induced protein abundance changes in human AM and autologous blood monocytes (MN). AM cells were activated by IFN-$\gamma$ stimulation resulting in STAT1 phosphorylation and production of MIG/CXCL9 chemokine. However, the global proteomic response to IFN-$\gamma$ in AM was dramatically limited in comparison to that of MN (9 AM vs 89 MN differentially abundant proteins). AM hypo-responsiveness was not explained by reduced JAK-STAT1 signaling nor increased SOCS1 expression. These findings suggest that AM have a tightly regulated response to IFN-$\gamma$ which may prevent excessive pulmonary inflammation but may also provide a niche for the initial survival and growth of Mtb and other intracellular pathogens in the lung.
NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia. H. H. L. Leung et al. Nature communications 2022 sep
Abstract
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of the adenoviral vector vaccines ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen) against COVID-19. The mechanisms involved in clot formation and thrombocytopenia in VITT are yet to be fully determined. Here we show neutrophils undergoing NETosis and confirm expression markers of NETs in VITT patients. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets, neutrophils, fibrin, extracellular DNA and citrullinated histone H3 in a flow microfluidics system and in vivo. Inhibition of NETosis prevents VITT-induced thrombosis in mice but not thrombocytopenia. In contrast, in vivo blockage of Fc$\gamma$RIIa abrogates both thrombosis and thrombocytopenia suggesting these are distinct processes. Our findings indicate that anti-PF4 antibodies activate blood cells via Fc$\gamma$RIIa and are responsible for thrombosis and thrombocytopenia in VITT. Future development of NETosis and Fc$\gamma$RIIa inhibitors are needed to treat VITT and similar immune thrombotic thrombocytopenia conditions more effectively, leading to better patient outcomes.
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