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 or mouse blood, the CD11b+Ly6G+ cell content of the enriched fraction is typically 88.2 ± 3.2% for bone marrow and 88.6 ± 4.9% for blood (mean ± SD) using the purple EasySep™ magnet. In the above example, the purities of the start and final enriched fractions are 46.6% and 89.1% (bone marrow) and 20.1% and 91.5% (blood), 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.
C6 peptide blockade of Hv1 channels inhibits neutrophil migration into the lungs to suppress Pseudomonas aeruginosa-induced acute lung injury
R. Zhao et al.
Respiratory Research 2025 Nov
Abstract
Background: Acute Lung Injury (ALI) and its most severe form, Acute Respiratory Distress Syndrome (ARDS), are critical pulmonary conditions characterized by life-threatening acute hypoxic respiratory failure, affecting over three million individuals globally each year. ALI involves alveolar inflammation and disruption of the alveolar-capillary barrier, primarily driven by neutrophil infiltration and the release of inflammatory mediators. In our previous study using a lipopolysaccharide (LPS)-induced mouse model of ALI, we demonstrated that C6, a peptide inhibitor of voltage-gated proton channels (Hv1), ameliorates lung injury, identifying Hv1 as a potential therapeutic target. However, (i) whether the anti-inflammatory effects of C6 are translatable to a clinically relevant live bacterial infection model, and (ii) the molecular mechanisms underlying these anti-inflammatory effects, remain unknown, and are a crucial next step towards targeted rational drug development. Methods: To induce ALI, we used an intratracheal Pseudomonas aeruginosa infection model, a gram-negative bacterium relevant in ventilated and immunocompromised patients. A separate group of infected mice also received intravenous treatment with C6 (4 mg/kg). Lung injury severity was evaluated using histopathological analysis. Bronchoalveolar lavage (BAL) fluid was collected to quantify neutrophil infiltration and proinflammatory cytokines concentrations. In addition, reactive oxygen species (ROS) production and intracellular calcium levels in BAL neutrophils were measured. RNA sequencing of BAL neutrophils was conducted to assess C6-induced transcriptional changes. Key findings were validated in vitro using human neutrophils. Results: C6 mitigates P. aeruginosa-induced ALI in mice by reducing neutrophil infiltration into the alveolar space by ~ 86%, improving lung injury scores, decreasing BAL fluid proinflammatory cytokine levels, and suppressing neutrophil ROS production and intracellular calcium levels. RNA sequencing of BAL neutrophils revealed 51 downregulated genes, including key regulators of neutrophil migration, cytokine release, and ROS production; only three genes were upregulated and they also have roles in neutrophil immune defense. In human neutrophils, C6 similarly inhibited chemotaxis and reduced ROS and cytokine release, and calcium influx. Conclusions: Targeting Hv1 with C6 effectively protects against P. aeruginosa-induced ALI by limiting neutrophil recruitment and activation. These findings establish C6 as a promising therapeutic candidate against infectious ALI and provide important mechanistic insights into its immunomodulatory effects on neutrophils.
Preceding influenza infection impacts neutrophil response to Aspergillus fumigatus and Staphylococcus aureus
N. Naghshtabrizi et al.
ImmunoHorizons 2025 Nov
Abstract
Influenza infection predisposes individuals to secondary pneumonia caused by a range of pathogens, including both bacterial and fungal organisms. Neutrophils are critical effector cells during infection. In this study, we analyzed the transcriptional pathways of lung neutrophils isolated from mouse models of influenza-associated pulmonary aspergillosis (IAPA) and post-influenza methicillin-resistant Staphylococcus aureus (MRSA) pneumonia to examine the immunopathological mechanisms underlying post-influenza super-infection. Pathways associated with neutrophil chemotaxis and degranulation were inhibited in IAPA compared to singular A. fumigatus infection and pathways associated with neutrophil recruitment and phagocytosis were inhibited in IAPA compared to singular influenza infection. Pathways associated with neutrophil recruitment and degranulation were inhibited in post-influenza MRSA pneumonia compared to singular MRSA infection and pathways associated with cytokine signaling were inhibited in post-influenza MRSA pneumonia compared to singular influenza infection. When the 2 types of super-infection were directly compared, pathways related to cytokine induction and neutrophil function were inhibited in IAPA neutrophils compared to post-influenza MRSA pneumonia. These data demonstrate that influenza causes neutrophil dysfunction, predisposing to secondary fungal and bacterial infections.
Heterogeneity of Neutrophils and Immunological Function in Neonatal Sepsis: Analysis of Molecular Subtypes Based on Hypoxia–Glycolysis–Lactylation
Mediators of Inflammation 2025 Mar
Abstract
Objective: Hypoxia–glycolysis–lactylation (HGL) may play a crucial role in neonatal sepsis (NS). This study aims to identify HGL marker genes in NS and explore immune microenvironment among NS subtypes. Materials and Methods: The gene expression dataset GSE69686, comprising 64 NS cases and 85 controls, was selected for analysis. Based on the screened HGL-related marker genes, diagnostic prediction models were constructed using nine machine learning algorithms, and molecular subtypes of NS were identified through consensus clustering. Subsequently, the heterogeneity of biological functions and immune cell infiltration among the different subtypes was analyzed. Finally, the marker genes and lactylation were validated using the GSE25504 dataset, clinical samples, and mouse neutrophil, respectively. Results: MERTK, HK3, PGK1, and STAT3 were identified and validated as marker genes, and the diagnostic prediction model for NS constructed using the support vector machine (SVM) algorithm exhibited optimal predictive performance. Based on gene expression patterns, two distinct NS subtypes were identified. Functional enrichment analysis highlighted significant immune-related pathways, while immune infiltration analysis revealed differences in neutrophil proportions between the subtypes. Furthermore, the expression levels of marker genes were positively correlated with neutrophil infiltration. Importantly, the experimental validation results were consistent with the findings from the bioinformatics analysis. Conclusion: This study identified the distinct NS subtypes and their associated marker genes. These findings will contribute to elucidating the disease's heterogeneity and establishing appropriate personalized therapeutic approaches.
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物种
小鼠
Magnet Compatibility
• EasySep™ Magnet (Catalog #18000) • “The Big Easy” EasySep™ Magnet (Catalog #18001) • RoboSep™-S (Catalog #21000)
EasySep™小鼠TIL（CD45）正选试剂盒
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