isolation kit cd63  (Miltenyi Biotec)

Journal: Current Issues in Molecular Biology

Article Title: Oral Squamous Cell Carcinoma Exosomes Upregulate PIK3/AKT, PTEN, and NOTCH Signaling Pathways in Normal Fibroblasts

doi: 10.3390/cimb47070568

Figure Lengend Snippet: Characterization of isolated exosomes: ( a ) NTA analysis (particles/mL), ( b ) BCA protein quantification, ( c ) purity ratio of samples, ( d ) representative WB image of CD9 and CD63 expression in exosomes isolated from SCC, DOK, and HaCaT cell lines, ( e ) quantification of CD9 protein expression in isolated exosomes, and ( f ) quantification of CD63 protein expression in isolated exosomes; ns—not significant.

Article Snippet: Exosome isolation with the MACS Exosome Isolation Kit CD63 (Miltenyi Biotec, Germany) was performed following the instructions of the manufacturer.

Techniques: Isolation, Expressing

Journal: bioRxiv

Article Title: Identification and proteomic profiling of CD90 + small EVs using a refined immunocapture separation approach targeting stromal-derived EV subpopulations in synovial fluid of arthritis patients

doi: 10.1101/2025.03.12.640750

Figure Lengend Snippet: (a) Graphical illustration of SEC-UF EV-isolation procedure. The supernatant of patient-derived synovial fibroblasts cultured for 48 h in Exo-depleted medium was centrifugated twice (500 x g 10 min, 3000 x g 10 min) to remove dead cells and cell debris. Cleared supernatant was ultrafiltered using a 100kDa cut-off Amicon column, before applying to the size exclusion chromatography (SEC) using an iZONE qEV1-35nm column. SEC fosters the division of particles regarding their size allowing to sample small EVs by collecting defined fractions. Fractions 5-9 were pooled and ultrafiltered/concentrated using a 10kDA cut-off Amicon column. (b) Representing TEM images of isolated small EVs derived from synovial fibroblasts cell culture (OA vs. RA) ranging from 30-130 nm. (c) Characterization of synovial fibroblast derived SEC-UF isolated EVs via Western Blot analysis. Loading of equal amount of lysed EV protein (4 μg) and corresponding cell lysate (2.5 μg) on a 4-15% SDS gradient gel and analyzed by Western Blot using specific antibodies detecting typical small EV markers (CD63, CD9, CD81, Syntenin-1, Annexin-1, Flotillin-1) and fibroblast markers (CD90, Podoplanin). Calnexin staining was added as EV negative control. (d) NanoFCM measurements of CD63, CD90 and Podoplanin antibody staining of synovial fibroblast cell culture derived EVs. Controls in the supplement (Supplement Figure 2). (e) Western Blot confirming membrane-associated location of CD90 and PDPN in lysates of fibroblasts and fibroblasts-derived EVs using MemPER-treatment separating the cytosolic (C) vs. membrane (M) protein fraction.

Article Snippet: For additional control experiments we used the Pan-separation KIT from Miltenyi (Mix: CD63, CD9, CD81, EV Isolation KIT Pan, human - 130-110-912).

Techniques: Isolation, Derivative Assay, Cell Culture, Size-exclusion Chromatography, Western Blot, Staining, Negative Control, Membrane

Journal: bioRxiv

Article Title: Identification and proteomic profiling of CD90 + small EVs using a refined immunocapture separation approach targeting stromal-derived EV subpopulations in synovial fluid of arthritis patients

doi: 10.1101/2025.03.12.640750

Figure Lengend Snippet: (a) Graphical illustration of immunocapture EV separation approach using prototype nano-sized magnetic beads (EV MicroBeads, Miltenyi). SEC-UF isolated EVs derived from supernatant of in vitro cultured synovial fibroblasts were incubated either with magnetic prototype MicroBeads coupled to anti-CD90 or anti-Podoplanin (PDPN) antibodies or an antibody isotype-coupled bead control (all provided by Miltenyi). Antibody-bound EVs (Marker + ) are retained by the magnetic column, while unbound EVs (Marker - ) are collected in the flowthrough (FT). A washing step (W) removed the remaining unbound/loosely associated EVs and protein aggregates. The removal of the magnet, released the bead-bound Marker + EVs, which were collected as Eluate (E1). The elution step was repeated (E2) to evaluate the efficacy of first elution step. (b) TEM measurement of eluate and flowthrough after CD90 and PDPN MicroBead separation. Arrows direct to EVs. (c) Western Blot analysis of CD90 and PDPN MicroBead separations and Isotype control. Detection of CD90, PDPN and typical EV marker (CD63, CD9, Syntenin-1, Annexin-1, Flotillin-1). EV Input (SEC-isolated EVs before separation) was loaded as control. (d) Quantification of CD90 and Podoplanin MicroBead separation from Western Blot results. Quantification of both PDPN populations (P1+P2) and only P2. Normalized to Syntenin-1 amount. 2way ANOVA, multiple comparisons, uncorrected Fishers’s LSD. (* p<0.05,** p<0.005, *** p<0.0005, **** p<0.0001)

Article Snippet: For additional control experiments we used the Pan-separation KIT from Miltenyi (Mix: CD63, CD9, CD81, EV Isolation KIT Pan, human - 130-110-912).

Techniques: Magnetic Beads, Isolation, Derivative Assay, In Vitro, Cell Culture, Incubation, Control, Marker, Western Blot