New format, same high quality! You may notice that your kit contents and packaging look slightly different from previous orders. We are currently updating the format of select EasySep™ Mouse kits to include a Mouse FcR blocker instead of Normal Rat Serum. With this change, all components will now be shipped in a single package, while providing the same cell isolation performance as before.
Starting with mouse bone marrow cells, the monocyte content (Lineage- (CD3, CD45R, CD117, CD49b, Siglec F) CD11b+Ly6G- Ly6Chi/lo) of the isolated fraction is 89.5 ± 4.8% (mean ± SD), using the purple EasySep™ Magnet. In the above example, monocyte purities in the start and final isolated fractions are 7.1% and 92.3%, respectively.
Figure 2. Data for Anti-Mouse CD11b Antibody, Clone M1/70, Alexa Fluor® 488-Conjugated
(A) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11b Antibody, Clone M1/70, Alexa Fluor® 488 (Catalog #60001AD) and anti-mouse CD45 APC. (B) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with a rat IgG2b, kappa Alexa Fluor® 488 isotype control antibody and anti-mouse CD45 APC. (C) Flow cytometry analysis of C57BL/6 mouse splenocytes processed with the EasySep™ Mouse Monocyte Enrichment Kit (Catalog #19861) and labeled with Anti-Mouse CD11b Antibody, Clone M1/70, Alexa Fluor® 488 (Catalog #60001AD). Histograms show labeling of splenocytes (Start) and isolated cells (Isolated). Labeling of start cells with a rat IgG2b, kappa Alexa Fluor® 488 isotype control antibody is shown in the bottom panel (open histogram). (D) Flow cytometry analysis of C57BL/6 mouse bone marrow cells processed with the EasySep™ Mouse Monocyte Enrichment Kit (Catalog #19861) and labeled with Anti-Mouse CD11b Antibody, Clone M1/70, Alexa Fluor® 488 (Catalog #60001AD). Histograms show labeling of bone marrow cells (Start) and isolated cells (Isolated). Labeling of start cells with a rat IgG2b, kappa Alexa Fluor® 488 isotype control antibody is shown in the bottom panel (open histogram).
Figure 3. Cell Isolation Protocol Lengths
Typical time taken (in minutes) to isolate cells using select EasySep™ kits.
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.
Interleukin-4 receptor alpha signaling regulates monocyte homeostasis. P. Haider et al. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2022 oct
Abstract
Interleukin-4 (IL-4) and its receptors (IL-4R) promote the proliferation and polarization of macrophages. However, it is unknown if IL-4R also influences monocyte homeostasis and if steady state IL-4 levels are sufficient to affect monocytes. Employing full IL-4 receptor alpha knockout mice (IL-4R$\alpha$-/- ) and mice with a myeloid-specific deletion of IL-4R$\alpha$ (IL-4R$\alpha$f/f LysMcre ), we show that IL-4 acts as a homeostatic factor regulating circulating monocyte numbers. In the absence of IL-4R$\alpha$, murine monocytes in blood were reduced by 50% without altering monocytopoiesis in the bone marrow. This reduction was accompanied by a decrease in monocyte-derived inflammatory cytokines in the plasma. RNA sequencing analysis and immunohistochemical staining of splenic monocytes revealed changes in mRNA and protein levels of anti-apoptotic factors including BIRC6 in IL-4R$\alpha$-/- knockout animals. Furthermore, assessment of monocyte lifespan in vivo measuring BrdU+ cells revealed that the lifespan of circulating monocytes was reduced by 55% in IL-4R$\alpha$-/- mice, whereas subcutaneously applied IL-4 prolonged it by 75%. Treatment of human monocytes with IL-4 reduced the amount of dying monocytes in vitro. Furthermore, IL-4 stimulation reduced the phosphorylation of proteins involved in the apoptosis pathway, including the phosphorylation of the NF$\kappa$Bp65 protein. In a cohort of human patients, serum IL-4 levels were significantly associated with monocyte counts. In a sterile peritonitis model, reduced monocyte counts resulted in an attenuated recruitment of monocytes upon inflammatory stimulation in IL-4R$\alpha$f/f LysMcre mice without changes in overall migratory function. Thus, we identified a homeostatic role of IL-4R$\alpha$ in regulating the lifespan of monocytes in vivo.
Monocytes transition to macrophages within the inflamed vasculature via monocyte CCR2 and endothelial TNFR2. V. Mysore et al. The Journal of experimental medicine 2022 may
Abstract
Monocytes undergo phenotypic and functional changes in response to inflammatory cues, but the molecular signals that drive different monocyte states remain largely undefined. We show that monocytes acquire macrophage markers upon glomerulonephritis and may be derived from CCR2+CX3CR1+ double-positive monocytes, which are preferentially recruited, dwell within glomerular capillaries, and acquire proinflammatory characteristics in the nephritic kidney. Mechanistically, the transition to immature macrophages begins within the vasculature and relies on CCR2 in circulating cells and TNFR2 in parenchymal cells, findings that are recapitulated in vitro with monocytes cocultured with TNF-TNFR2-activated endothelial cells generating CCR2 ligands. Single-cell RNA sequencing of cocultures defines a CCR2-dependent monocyte differentiation path associated with the acquisition of immune effector functions and generation of CCR2 ligands. Immature macrophages are detected in the urine of lupus nephritis patients, and their frequency correlates with clinical disease. In conclusion, CCR2-dependent functional specialization of monocytes into macrophages begins within the TNF-TNFR2-activated vasculature and may establish a CCR2-based autocrine, feed-forward loop that amplifies renal inflammation.
IL-10 Dysregulation Underlies Chemokine Insufficiency, Delayed Macrophage Response, and Impaired Healing in Diabetic Wounds. R. Roy et al. The Journal of investigative dermatology 2022 mar
Abstract
Persistent inflammation is a major contributor to healing impairment in diabetic chronic wounds. Paradoxically, diabetic wound environment during the acute phase of healing is completely different because it exhibits a reduced macrophage response owing to inadequate expression of CCL2 proinflammatory cytokine. What causes a reduction in CCL2 expression in diabetic wounds early after injury remains unknown. In this study, we report that in contrast to prolonged exposure to high glucose, which makes monocytes proinflammatory, short-term exposure to high glucose causes a rapid monocyte reprogramming, manifested by increased expression and secretion of IL-10, which in an autocrine/paracrine fashion reduces glucose uptake and transforms monocytes into an anti-inflammatory phenotype by dampening signaling through toll-like receptors. We show that IL-10 expression is significantly increased in diabetic wounds during the acute phase of healing, causing significant reductions in toll-like receptor signaling and proinflammatory cytokine production, delaying macrophage and leukocyte responses, and underlying healing impairment in diabetic wounds. Importantly, blocking IL-10 signaling during the acute phase of healing improves toll-like receptor signaling, increases proinflammatory cytokine production, enhances macrophage and leukocyte responses, and stimulates healing in diabetic wounds. We posit that anti-IL-10 strategies have therapeutic potential if added topically after surgical debridement, which resets chronic wounds into acute fresh wounds.
Thank you for your interest in IntestiCult™ Organoid Growth Medium (Human). Please provide us with your contact information and your local representative will contact you with a customized quote. Where appropriate, they
can also assist you with a(n):
Estimated delivery time for your area
Product sample or exclusive offer
In-lab demonstration
By submitting this form, you are providing your consent to STEMCELL Technologies Canada Inc. and its subsidiaries and affiliates (“STEMCELL”) to collect and use your information, and send you newsletters and emails in accordance with our
privacy policy. Please contact us with any questions that you may have. You can unsubscribe or change your email preferences at any time.
PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT STEMCELL, REFER TO WWW.STEMCELL.COM/COMPLIANCE.
