Isolate highly purified B cells directly from human whole blood without the need for density gradient centrifugation, RBC sedimentation or lysis. This platform provides fast and fully immunomagnetic negative selection.
This cell isolation kit targets non-B cells for removal with EasySep™ Direct RapidSpheres™ and antibodies recognizing specific surface antigens. Unwanted cells are separated from desired cells using an EasySep™ magnet, and untouched target cells are then pipetted or poured into a new tube.
Isolated cells can be used directly in downstream applications such as flow cytometry, culture or DNA/RNA extraction.
Figure 1. Typical EasySep™ Direct Human B Cell Isolation Profile
Starting with human whole blood from normal healthy donors, the typical B cell (CD3-CD19+) content of the non-lysed final isolated fraction is 95.3 ± 2.7% (gated on CD45) or 88.5 ± 11.5% (not gated on CD45). In the above example, the B cell (CD3-CD19+) content of the lysed whole blood start sample and the non-lysed final isolated fraction is 6.2% and 95.9% (gated on CD45), respectively, or 6.2% and 95.8% (not gated on CD45), respectively. The starting frequency of B cells in the non-lysed whole blood start sample above is 0.011% (data not shown).
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.
Raman-based spectrophenotyping of the most important cells of the immune system. A. Borek-Dorosz et al. Journal of advanced research 2022 nov
Abstract
INTRODUCTION Human peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of cells that includes T and B lymphocytes. The total number of lymphocytes and their percentage in the blood can be a marker for the diagnosis of several human diseases. Currently, cytometric methods are widely used to distinguish subtypes of leukocytes and quantify their number. These techniques use cell immunophenotyping, which is limited by the number of fluorochrome-labeled antibodies that can be applied simultaneously. OBJECTIVE B and T lymphocytes were isolated from peripheral blood obtained from healthy human donors. METHODS The immunomagnetic negative selection was used for the enrichment of B and T cells fractions, and their purity was assessed by flow cytometry. Isolated cells were fixed with 0.5% glutaraldehyde and measured using confocal Raman imaging. K-means cluster analysis, principal component analysis and partial least squares discriminant methods were applied for the identification of spectroscopic markers to distinguish B and T cells. HPLC was the reference method for identifying carotene in T cells. RESULTS Reliable discrimination between T and B lymphocytes based on their spectral profile has been demonstrated using label-free Raman imaging and chemometric analysis. The presence of carotene in T lymphocytes (in addition to the previously reported in plasma) was confirmed and for the first time unequivocally identified as $\beta$-carotene. In addition, the molecular features of the lymphocytes nuclei were found to support the discriminant analysis. It has been shown that although the presence of carotenoids in T cells depends on individual donor variability, the reliable differentiation between lymphocytes is possible based on Raman spectra collected from individual cells. CONCLUSIONS This proves the potential of Raman spectroscopy in clinical diagnostics to automatically differentiate between cells that are an important component of our immune system.
Cardiovascular risk factors: The effects of ageing and smoking on the immune system, an observational clinical study. H. W. Grievink et al. Frontiers in immunology 2022
Abstract
Currently immunomodulatory compounds are under investigation for use in patients with cardiovascular disease, caused by atherosclerosis. These trials, using recurrent cardiovascular events as endpoint, require enrollment of large patient groups. We investigated the effect of key risk factors for atherosclerosis development, ageing and smoking, on the immune system, with the objective to identify biomarkers differentiating between human populations, and potentially serving as endpoints for future phase 1B trials with immunomodulatory compounds. Blood was collected from young healthy volunteers (aged 18-25 years, n=30), young smokers (18-25 years, n=20), elderly healthy volunteers (>60 years, n=20), heavy smokers (>45 years, 15 packyears, n=11) and patients with stable coronary artery disease (CAD) (>60 years, n=27). Circulating immune cell subsets were characterized by flow cytometry, and collected plasma was evaluated by proteomics (Olink). Clear ageing effects were observed, mostly illustrated by a lower level in CD8+ and na{\{i}}ve CD4+ and CD8+ T cells with an increase in CD4+ and CD8+ effector memory T cells in elderly healthy volunteers compared to young healthy volunteers. Heavy smokers showed a more inflammatory cellular phenotype especially a shift in Th1/Th2 ratio: higher Th1 and lower Th2 percentages compared to young healthy volunteers. A significant decrease in circulating atheroprotective oxLDL-specific IgM was found in patients with CAD compared to young healthy volunteers. Elevated pro-inflammatory and chemotactic proteins TREM1 and CCL11 were observed in elderly volunteers compared to young volunteers. In addition heavy smokers had an increase in pro-inflammatory cytokine IL-6 and lysosomal protein LAMP3. These data show that ageing and smoking are associated with an inflammatory immunophenotype and that heavy smokers or aged individuals may serve as potential populations for future clinical trials investigating immunomodulatory drugs targeted for cardiovascular disease."
A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells. Xiao X et al. mAbs 2016 JUL
Abstract
Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool.
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