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Figure 1. Typical EasySep? Direct Protocol

Figure 2. Typical EasySep? Direct Human PBMC Isolation Profile

In the above example, the mononuclear cell content of the whole blood start sample (lysed by ammonium chloride) and non-lysed final isolated fraction is 27.0% and 98.6% (not gated on CD45), respectively.

Figure 3. Representative Flow Cytometry Plots

Representative Forward Scatter (FSC) vs. Side Scatter (SSC) flow cytometry plots of mononuclear cells isolated from whole blood samples using either density gradient centrifugation or EasySep? Direct Human PBMC Isolation Kit.

Figure 4. Representative t-SNE plots of Isolated PBMCs

Representative t-SNE plots of PBMCs stained with 19 markers and analyzed with mass cytometry (CyTOF). Cells are clustered and colored based on the combination of markers they express. Both density gradient centrifugation and EasySep? Direct Human PBMC Isolation Kit resulted in comparable cell populations with the exception of the contaminating granulocyte population (CD16+CD15+CD11b+CD66b+ CD45low).

Figure 5. EasySep? Direct Human PBMC Isolation Kit Results in Fewer Contaminating Cells Compared to Density Gradient Centrifugation

Mononuclear cells were isolated from whole blood samples using either density gradient centrifugation or EasySep? Direct Human PBMC Isolation Kit. Cells were counted and analyzed by flow cytometry. (A) Both density gradient centrifugation and EasySep? Direct Human PBMC Isolation Kit resulted in an equivalent total number of nucleated cells recovered from 24-hour blood samples (mean ¡À SD; n=14). (B) Using EasySep? Direct Human PBMC Isolation Kit to obtain mononuclear cells from 24-hour old blood samples resulted in lower numbers of residual platelets (CD41+), red blood cells (Glycophorin A+/CD45-), and granulocytes (CD66b+) compared to density gradient centrifugation (mean ¡À SD; n=15). (C) Cell isolation from 24-hour, 48-hour, 72-hour and 96-hour old blood samples using EasySep? Direct Human PBMC Isolation Kit resulted in lower numbers of residual granulocytes compared to cell isolation using density gradient centrifugation (mean ¡À SD; n=3).

Figure 6. PBMCs Isolated with EasySep? Direct Human PBMC Isolation Kit Proliferate and Maintain High RNA Integrity

Mononuclear cells were isolated from whole blood samples using either density gradient centrifugation or EasySep? Direct Human PBMC Isolation Kit. (A) Isolated mononuclear cells were used for downstream RNA isolation. There was no significant difference in RNA integrity as measured with the Agilent RNA Bioanalyzer (mean ¡À SEM, n=3). (B) Isolated mononuclear cells were labeled with Proliferation Dye eFluor 450 and stimulated with ImmunoCult? Human CD3/CD28 T Cell Activator and 0.5ng/ml IL-2. After 4 days in culture, cells were analyzed for proliferation by flow cytometry. Representative histogram showing dividing cells (eFluor 450low).

Figure 7. PBMC Isolation from a Full-Size Leukapheresis Pack Using the EasySep? Direct Human PBMC Isolation Kit Automated with RoboSep?-S Results in Fewer Contaminating Cells Compared to Density Gradient Centrifugation

Mononuclear cells were isolated from single concentrated leukapheresis packs using either density gradient centrifugation with Lymphoprep? density gradient medium (Density Gradient Centrifugation) or EasySep? Direct Human PBMC Isolation Kit automated on the RoboSep?-S instrument (EasySep? Direct on RoboSep?-S). Cells were counted and analyzed by flow cytometry. Compared to density gradient centrifugation, EasySep? Direct Human PBMC Isolation Kit automated on the RoboSep?-S instrument resulted in (A) equivalent numbers of total mononuclear cells, (B) equivalent numbers of total monocytes and lymphocytes and (C) lower numbers of residual platelets (CD41+), red blood cells (Glycophorin A+ /CD45? ), and granulocytes (CD66b+ ) (mean ¡À SD n=6).

Figure 8. Easy 250 EasySep? Magnet Can Be Used to Isolate Mononuclear Cells from Whole Blood and Leukopak Samples of Up to 125 mL

Figure 9. EasySep? Direct Human PBMC Isolation Kit Reduces Platelet Contamination More Efficiently Than Column-Based Methods

Peripheral blood mononuclear cells (PBMCs) were isolated from 24-hour-old whole blood samples using either the EasySep? Direct Human PBMC Isolation Kit or alternative, column-based kits. Flow cytometry analysis demonstrated that PBMCs isolated with the EasySep? kit contained significantly fewer contaminating platelets (CD42b+), while levels of residual red blood cells (Glycophorin A+/CD45-) and granulocytes (CD66b+) were comparable between methods. Data are presented as mean ¡À SD (n ¡Ý 3 per condition).

Figure 10. EasySep? Direct PBMC Isolation Kit Achieves Higher Recovery and Purity of Mononuclear Cells Compared to Column-Based Kits

PBMCs were isolated from 24-hour-old whole blood samples using the EasySep? Direct Human PBMC Isolation Kit and two column-based kits designed for PBMC isolation from whole blood. Isolated cells were labeled with anti-CD45 antibodies and analyzed by flow cytometry. Cell purity was assessed by the proportion of CD45+ events among total recorded events (serving as an indicator of debris), and total recovery was quantified across isolation methods. The EasySep? Direct PBMC Isolation Kit yielded higher purity PBMCs than both Column-Based Kits A and B. It also recovered a greater number of CD45+ mononuclear cells compared to Column-Based Kit A, with a comparable recovery to Column-Based Kit B. Data are presented as mean ¡À SD (n ¡Ý 3 per condition).