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.
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.
Cutting Edge: Mycobacterium tuberculosis Induces Aerobic Glycolysis in Human Alveolar Macrophages That Is Required for Control of Intracellular Bacillary Replication. Gleeson LE et al.
Journal of Immunology 2016 MAR
Abstract
Recent advances in immunometabolism link metabolic changes in stimulated macrophages to production of IL-1β,a crucial cytokine in the innate immune response to Mycobacterium tuberculosis. To investigate this pathway in the host response to M. tuberculosis,we performed metabolic and functional studies on human alveolar macrophages,human monocyte-derived macrophages,and murine bone marrow-derived macrophages following infection with the bacillus in vitro. M. tuberculosis infection induced a shift from oxidative phosphorylation to aerobic glycolysis in macrophages. Inhibition of this shift resulted in decreased levels of proinflammatory IL-1β and decreased transcription of PTGS2,increased levels of anti-inflammatory IL-10,and increased intracellular bacillary survival. Blockade or absence of IL-1R negated the impact of aerobic glycolysis on intracellular bacillary survival,demonstrating that infection-induced glycolysis limits M. tuberculosis survival in macrophages through induction of IL-1β. Drugs that manipulate host metabolism may be exploited as adjuvants for future therapeutic and vaccination strategies.
FNDC4 acts as an anti-inflammatory factor on macrophages and improves colitis in mice. Bosma M et al.
Nature Communications 2016 APR
Abstract
FNDC4 is a secreted factor sharing high homology with the exercise-associated myokine irisin (FNDC5). Here we report that Fndc4 is robustly upregulated in several mouse models of inflammation as well as in human inflammatory conditions. Specifically,FNDC4 levels are increased locally at inflamed sites of the intestine of inflammatory bowel disease patients. Interestingly,administration of recombinant FNDC4 in the mouse model of induced colitis markedly reduces disease severity compared with mice injected with a control protein. Conversely,mice lacking Fndc4 develop more severe colitis. Analysis of binding of FNDC4 to different immune cell types reveals strong and specific binding to macrophages and monocytes. FNDC4 treatment of bone marrow-derived macrophages in vitro results in reduced phagocytosis,increased cell survival and reduced proinflammatory chemokine expression. Hence,treatment with FNDC4 results in a state of dampened macrophage activity,while enhancing their survival. Thus,we have characterized FNDC4 as a factor with direct therapeutic potential in inflammatory bowel disease and possibly other inflammatory diseases.
Therapeutic Blockade of Immune Complex-Mediated Glomerulonephritis by Highly Selective Inhibition of Bruton's Tyrosine Kinase. Chalmers SA et al.
Scientific Reports 2016 MAY
Abstract
Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of lupus patients. Bruton's tyrosine kinase (BTK) is important for B cell development,Fc receptor signaling,and macrophage polarization. In this study,we investigated the effects of a novel,highly selective and potent BTK inhibitor,BI-BTK-1,in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1,either prophylactically or therapeutically. When compared with control treated mice,NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated kidney disease,which was dose dependent. BI-BTK-1 treatment resulted in decreased infiltrating IBA-1+ cells,as well as C3 deposition within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly,when administered therapeutically,BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis,and BTK inhibition as a promising therapeutic target for LN.
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