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Figure 1. EasySep™ Human Extracellular Vesicle (CD61) Depletion and Positive Selection Kit Efficiently Depletes CD61+ Extracellular Vesicles from Human Plasma

Plasma from normal, healthy donors was processed and CD61+ extracellular vesicles (EVs) were depleted using the EasySep™ Human Extracellular Vesicle (CD61) Depletion Protocol (see Product Information Sheet). EVs were then isolated from 200 µL of CD61-depleted plasma and control plasma (no depletion) using the EasySep™ Human Pan-Extracellular Vesicle Positive Selection Kit. Isolated EVs were resuspended in 20 µL, and 9 µL was used per sample for western blot analysis. Western blot analysis was performed under non-reducing conditions to assess the recovery of CD61 (the target marker), CD9 and CD63 (markers expressed on both platelet-derived and non-platelet-derived EVs), and CD81 (only expressed on non-platelet-derived EVs). Quantification of CD61 signal intensity from the same image at the same exposure time for the depleted and control plasma revealed that, on average, 94.7 ± 3.5% (mean ± SD) of CD61+ EVs were depleted compared to the control (using the purple EasySep™ Magnet). Platelet-derived EVs are expected to be CD61+, CD63+, CD9+, and CD81- (Kholia et al., 2016; Malys et al., 2023). After CD61 depletion, other markers expressed on platelet-derived EVs (CD63 and CD9) were also reduced, while markers only expressed on non-platelet EVs (CD81) were maintained. In the above example, 95% of CD61+ EVs were depleted compared to the control.

Figure 2. Differential Ultracentrifugation-Purified EVs Can Be Successfully Depleted Using the EasySep™ Human Extracellular Vesicle (CD61) Depletion Protocol

CD61+ extracellular vesicles were depleted from differential ultracentrifugation-purified EV preparations derived from human plasma of healthy donors using the EasySep™ Human Extracellular Vesicle (CD61) Depletion Protocol. A 9 µL fraction from depleted samples were compared to the same volume of non-depleted controls. CD61 and CD81 were analyzed by western blot under non-reducing conditions. Quantification of CD61 intensity from the same image at the same exposure time for both samples revealed that, on average, 95.7± 4.9% (mean ± SD) of CD61+ EVs were depleted compared to the control (using the purple EasySep™ Magnet). In the above example, starting with a 1 mL differential ultracentrifugation-purified EV preparation with 8.6 x 10¹⁰ extracellular particles, 85% of CD61+ EVs and 5.2 x 10¹⁰ extracellular particles were depleted based on western blot and Spectradyne nCS1 analysis (size range 72 - 400 nm, transit time < 100 µs), respectively.

Figure 3. CD61+ EVs Can Be Depleted from Human Plasma with the EasySep™ Human Extracellular Vesicle (CD61) Depletion Protocol, Followed by Downstream EV Size Exclusion Chromatography (SEC) EV Purification

CD61+ EVs from processed human plasma samples from normal healthy donors were depleted using the EasySep™ Human Extracellular Vesicle (CD61) Depletion Protocol. EVs from 1 mL of CD61-depleted plasma and control plasma (no depletion) were subsequently isolated using the 2 mL Extracellular Vesicle SEC Columns. 500 µL of each fraction were collected and analyzed. Both the CD61 depleted plasma and the control plasma showed the expected extracellular vesicle (EV) elution profile. (A) CD61, CD9, CD63, and CD81 were analyzed by western blot under non-reducing conditions. In the above example, CD61+ EVs were not detected in fractions 7 - 9 of the CD61 depleted plasma but were readily detected in fractions 7 - 9 of the control (no depletion) plasma. (B) Extracellular particle concentrations were measured by Spectradyne nCS1 (size range 72 - 400 nm, transit time <100 µs) and protein concentration was measured by BCA assay. Extracellular particles were detected in fractions 6 - 9 and proteins appeared in fractions 10 - 11 onward. A slight shift in elution fractions was observed, likely due to variability in manual fraction collection. In this example, 5.1 × 10⁹ extracellular particles, or 42% of the total extracellular particles, were depleted from the CD61-depleted plasma compared to the control.

Figure 4. CD61+ EV Depletion Improves the Ability to Detect Non-Platelet-Derived EVs

Starting with differential ultracentrifugation-purified EV preparations from human plasma of normal healthy donors, CD61+ EVs were depleted using the EasySep™ Human Extracellular Vesicle (CD61) Depletion Protocol and compared to control (no depletion). Extracellular particle (EP) concentrations from EV preparations before and after CD61 depletion were measured by Spectradyne nCS1 (size range 72 - 400 nm, transit time <100 µs). For western blot analysis, 1.3 × 10¹⁰ EPs were loaded per well. (A) CD61 and CD81 were analyzed by western blot under non-reducing conditions. (B) CD61 and CD81 signal intensities were quantified from the same image at the same exposure time for both samples before and after CD61 depletion. The signal-to-noise ratio was defined as the CD81 intensity relative to the CD61 intensity of the corresponding sample. In the above example, the signal-to-noise ratio improved from 0.2 to 4.4 after CD61 depletion.

Figure 5. Selective Isolation of Platelet-Derived EVs Using the EasySep™ Human Extracellular Vesicle (CD61) Positive Selection Protocol

Processed human plasma samples from normal healthy donors were positively selected using either the EasySep™ Human Extracellular Vesicle (CD61) Positive Selection Protocol or the EasySep™ Human Pan-Extracellular Vesicle Positive Selection Kit. CD61, CD9, CD63, and CD81 were analyzed by western blot under non-reducing conditions. In the above example, the CD61 positively selected EVs demonstrated the expected phenotype of platelet-derived EVs (CD61+, CD63+, CD9+, and CD81-) (Kholia et al., 2016; Malys et al., 2023).

Figure 6. Sequential Isolation of CD61+ EV from Plasma and Subsequent Recapture of Remaining EVs Can Be Achieved Using the EasySep™ Human Extracellular Vesicle (CD61) Depletion and Positive Selection Protocol

1 mL of processed plasma was either depleted using the EasySep™ Human Extracellular Vesicle (CD61) Depletion and Positive Selection Protocol* or left untreated as a control. Following depletion, the plasma was poured off into a new tube, while the remaining fraction in the tube was magnetically washed to recover the CD61+ EVs. The remaining EVs in the depleted (or control) plasma were then recaptured using the EasySep™ Human Pan-Extracellular Vesicle Positive Selection Kit (n = 2). In the above example, the CD61 positively selected EVs displayed the expected phenotype of platelet-derived EVs (CD61+, CD63+, CD9+, and CD81-). The remaining EVs recaptured after CD61 depletion were CD61-, CD63+, CD9+, and CD81+. In contrast, the remaining EVs isolated from the control plasma, which represented all EVs in the sample, were positive for all markers analyzed. The CD61 positive selection recovered 125% of CD61+ EVs compared to the control sample EVs, which was within the normal experiment variability. The remaining EVs captured after CD61 depletion showed a 94% reduction in CD61+ EVs and a 104% recovery of CD81+ EVs compared to the control sample.

*Not included in the Product Information Sheet. Please contact Product and Scientific Support for the combined depletion and positive selection protocol.