optiprep tm gradient fractions  (Bio-Rad)

Journal: Scientific Reports

Article Title: Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection

doi: 10.1038/srep24316

Figure Lengend Snippet: ( A , B ) Quantification of FCM data and Western-blotting of PLT concentrate-derived MV fractions purified on an Optiprep TM density gradient (n = 3, mean + SEM). The event number was detected within the MV-gate. MVs were detected by AX (FCM) and CD63 (Western blotting) ( A ) and lipoproteins by apoB (550 kDa) ( B ). Of note, Western blotting only shows one of the analyzed samples while the FCM shows the average ± SEM of 3 measurements. ( C ) Comparison of the apoB-positive events (black bars) and the AX-positives (gray bars) (FCM, n = 2, mean + SEM). ( D ) SEC purification of MVs isolated from PLT concentrates, fractions analyzed by FCM. (apoB: black; AX: gray; CD41a: light gray bars, n = 2, mean + SEM) Note that the apoB-positivity was co-purified with the EV markers.

Article Snippet: Optiprep TM gradient fractions with identical volumes were pelleted, resuspended in lysis buffer, and electrophoresed in agarose-SDS gel at constant 100 V for 5 h. Proteins were blotted to PVDF membranes (Bio-Rad).

Techniques: Western Blot, Derivative Assay, Purification, Comparison, Isolation

Journal: Scientific Reports

Article Title: Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection

doi: 10.1038/srep24316

Figure Lengend Snippet: ( A , B ) FCM detection of the indicated markers in EXOs conjugated onto latex beads. The EXOs were isolated from fasting PFP ( A ) or PLT concentrate ( B ) by differential UC and gravity size filtration (gray histograms: blocked beads incubated with antibody, empty histograms: EXO sample). ( C ) EXOs from fasting PFP purified on an Optiprep TM density-gradient. Distribution of CD9 positive events was determined by FCM (upper panel, n = 3, mean + SEM) and CD63 positivity of fractions was determined by Western blotting (lower panel). Of note, Western blotting only shows one of the analyzed samples while the FCM shows the average ± SEM of 3 measurements. ( D ) The distribution of apoB-positive events determined by FCM (upper panel, n = 3) and by Western blotting (lower panel) from the same samples as in ( C ). ( E ) PLT-derived EXOs were also purified on a density-gradient. The EXO containing FR7-8 was analyzed by FCM for CD9-, CD63- and apoB-positivity (gray histograms: blocked beads incubated with antibody, empty histograms: EXO sample).

Article Snippet: Optiprep TM gradient fractions with identical volumes were pelleted, resuspended in lysis buffer, and electrophoresed in agarose-SDS gel at constant 100 V for 5 h. Proteins were blotted to PVDF membranes (Bio-Rad).

Techniques: Isolation, Filtration, Incubation, Purification, Western Blot, Derivative Assay