optiprep gradient fractionation  (Progen Biotechnik)

Journal: Cancer discovery

Article Title: Pharmacological suppression of B7-H4 glycosylation restores antitumor immunity in immune-cold breast cancers

doi: 10.1158/2159-8290.CD-20-0402

Figure Lengend Snippet: (A) SKBR3 cells were treated with 10 μM doxorubicin and/or 10 μM NGI-1 for 24 h. Membrane CALR, HSP70 and HSP90 were measured by flow cytometry. (B) MDA-MB-468-vector and MDA-MB-468-B7-H4 knockout cells were established and treated with 5 μM doxorubicin for 24 h. Immunofluorescence staining of the immunogenic cell death markers CALR on the cell surface was performed. Mean fluorescence index of CALR was quantified by ImageJ. Representative images are shown. (C-D) SKBR3, MDA-MB-468, MDA-MB-468-vector, MDA-MB-468-B7-H4 knockout cells were treated with 1 or 10 μM doxorubicin and/or 10 μM NGI-1 for 24 h. p-eIF2a and actin were examined by immunoblotting. Scale bar, 100 μm. (E) Representative paired immunohistochemistry staining of B7-H4 and phospho eIF2α (Ser51) in tissue array BC081120. Statistical analysis of immunohistochemical staining indicates B7-H4 expression is negatively correlated with p-eIF2α expression in breast cancer (r = −0.249, p =8.71x10−3). (F) MDA-MB-468-Flag-hB7-H4 were treated in the presence or absence of doxorubicin (10 μM) and/or NGI-1 (10 μM). Then Flag-hB7-H4 was immunoprecipitated followed by immunoblot. The indicated proteins were examined. (G) Schematic diagram of the procedure of OptiPrep density gradient assay with 24 collected fractions from low to high density is shown. MDA-MB-468-vector and MDA-MB-468-hB7-H4 knockout cells were treated with 10 μM doxorubicin for 24 hr followed by OptiPrep density gradient assay. HSP90, CALR, eIF2α and p-eIF2α in fraction 1 to 13 were examined by immunoblotting. (H) eIF2a was immunoprecipitated in fraction 13 in both MDA-MB-468-vector and MDA-MB-468-B7-H4 knockout cells followed by immunoblotting. PERK, eIF2α and p-eIF2α were examined.

Article Snippet: OptiPrep density gradient protein fractionation assay To prepare the iodixanol gradient, a 50% (w/v) and 5% (w/v) solution of iodixanol were made by diluting the stock solution (60% (w/v) aqueous iodixanol from StemCell Technologies with 0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl pH7.4.

Techniques: Flow Cytometry, Plasmid Preparation, Knock-Out, Immunofluorescence, Staining, Fluorescence, Western Blot, Immunohistochemistry, Immunohistochemical staining, Expressing, Immunoprecipitation

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