rabbit anti-mouse cd9  (ZenBio)

Journal: International Journal of Nanomedicine

Article Title: An Isolation System to Collect High Quality and Purity Extracellular Vesicles from Serum

doi: 10.2147/IJN.S328325

Figure Lengend Snippet: Procedures and characteristics of SEC for separating EVs from serum. ( A ) Schematic overview of the experimental workflow. ( B ) Concentrations of particles and proteins in the SEC fractions were determined with nanoparticle tracking analysis (gray) and BCA (red), respectively. Data shows all 24 fractions. ( C ) SDS-PAGE was used to determine to directly visualize the relative presence of proteins in the all collected fractions, 15 μL of each fraction was mixed with 5 μL 4-fold concentrated reducing sample buffer, boiled for 5 minutes, and loaded on a 10% gradient gel. ( D ) The presence of the vesicle marker CD9, CD81, and the serum contaminant protein albumin and apolipoprotein marker apoA were determined in pooled EVs concentrates of fractions 8–13 and pooled Non-EVs concentrates fractions 14–24 with Western blot. ( E ) Droplets of fractions 9–11 and 20 were loaded onto grids, negative stained, and evaluated with transmission electron microscopy (TEM). Examples of EV-like structures (cup-shaped) are indicated by white arrows. Scale bars are 200 nm.

Article Snippet: CD9 was detected using rabbit anti-mouse CD9 (220642, Zen Bio, 1:1000); CD63 with rabbit anti-mouse CD63 (510953, Zen Bio, 1:1000); CD81 with rabbit anti-mouse CD81 (381296, Zen Bio, 1:1000); apoA1 with rabbit anti-mouse apoA1 (381145, Zen Bio, 1:1000); Albumin with rabbit anti-mouse Albumin (ab207327, Abcam, 1:1000).

Techniques: SDS Page, Marker, Western Blot, Staining, Transmission Assay, Electron Microscopy

Journal: International Journal of Nanomedicine

Article Title: An Isolation System to Collect High Quality and Purity Extracellular Vesicles from Serum

doi: 10.2147/IJN.S328325

Figure Lengend Snippet: Systematically compare the separation efficiency of SEC, UC, and TEI. ( A ) Schematic overview of the experimental workflow. ( B ) total protein was determined by BCA (expressed as mg/mL originating serum; mean ± SD, n = 3). ( C ) the concentration of particles as detected by NTA (particles/mL originating serum; mean ± SD, n = 3). ( D ) the ratio of particle to protein for SEC, UC, and TEI. ( E ) size distribution of particles detected in C (representative for n = 3). The particle size at peak optimum is indicated. ( F ) samples were analyzed by SDS-PAGE followed by Bio-Safe Coomassie G-250. ( G ) the same samples were analyzed by Western blotting for the presence of the EV markers CD9, CD81, CD63, and the serum contaminant protein albumin and apolipoprotein marker apoA. The experiment shown is representative of 3 independent experiments. ( F and G ) SEC, TEI, and UC samples are all separated from equivalent volumes of 1mL originating serum, and the final volume is 100 μL. UCx3 samples were separated from 3mL originating serum, and the final volume is 100 μL. All samples were loaded on equal volumes for electrophoresis. ( H ) The whole amount of TEM images of particles collected by SEC, UC, or TEI. Examples of EV-like structures (cup-shaped) are indicated by white arrows. Scale bars are 500 nm. Data were used for comparative analysis and presented as columns with bars representing means ± SD. Marks: *p≤0.05; **p≤0.01; ***p≤0.001 and non-significant differences were indicated using ns symbol.

Article Snippet: CD9 was detected using rabbit anti-mouse CD9 (220642, Zen Bio, 1:1000); CD63 with rabbit anti-mouse CD63 (510953, Zen Bio, 1:1000); CD81 with rabbit anti-mouse CD81 (381296, Zen Bio, 1:1000); apoA1 with rabbit anti-mouse apoA1 (381145, Zen Bio, 1:1000); Albumin with rabbit anti-mouse Albumin (ab207327, Abcam, 1:1000).

Techniques: Concentration Assay, SDS Page, Western Blot, Marker, Electrophoresis

Journal: International Journal of Nanomedicine

Article Title: An Isolation System to Collect High Quality and Purity Extracellular Vesicles from Serum

doi: 10.2147/IJN.S328325

Figure Lengend Snippet: Comparison of two different combination methods and SEC. ( A ) Schematic overview of the experimental workflow. ( B ) total protein was determined by BCA (expressed as mg/mL originating serum; mean ± SD, n = 3). ( C ) the concentration of particles as detected by NTA (particles/mL originating serum; mean ± SD, n = 3). ( D ) the ratio of particle to protein for SEC, SEC+UC, and SEC+TEI. ( E ) size distribution of particles detected in C (representative for n = 3). The particle size at peak optimum is indicated. ( F ) samples were analyzed by SDS-PAGE followed by Bio-Safe Coomassie G-250. ( G ) the same samples were analyzed by Western blotting for the presence of the EV markers CD9, CD81, CD63, and the serum contaminant protein albumin and apolipoprotein marker apoA. The experiment shown is representative of 3 independent experiments. ( F and G ) SEC, SEC+UC, and SEC+TEI samples are all separated from equivalent volumes of 1mL originating serum, and the final volume is 100 μL. SEC+UCx3 samples were separated from 3mL originating serum, and the final volume is 100μL. All samples were loaded on equal volumes for electrophoresis. ( H ) The whole amount of TEM images of particles collected by SEC, SEC+UC, or SEC+TEI. Examples of EV-like structures (cup-shaped) are indicated by white arrows. Scale bars are 500 nm. Data were used for comparative analysis and presented as columns with bars representing means ± SD. Marks: *p≤0.05; **p≤0.01; ***p ≤ 0.001 and non-significant differences were indicated using ns symbol.

Article Snippet: CD9 was detected using rabbit anti-mouse CD9 (220642, Zen Bio, 1:1000); CD63 with rabbit anti-mouse CD63 (510953, Zen Bio, 1:1000); CD81 with rabbit anti-mouse CD81 (381296, Zen Bio, 1:1000); apoA1 with rabbit anti-mouse apoA1 (381145, Zen Bio, 1:1000); Albumin with rabbit anti-mouse Albumin (ab207327, Abcam, 1:1000).

Techniques: Comparison, Concentration Assay, SDS Page, Western Blot, Marker, Electrophoresis

Journal: International Journal of Molecular Sciences

Article Title: Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

doi: 10.3390/ijms22041672

Figure Lengend Snippet: Cyclophosphamide significantly decreased the testicular weight and seminiferous tubule normal histology, VASA cells GFR-α-1, α-6-Integrin, CD9, and C-KIT cells counts in the tubules of immature mice: cyclophosphamide (CP) was intraperitoneally injected (i.p; 100 mg/kg in 100 uL; see methodology section) (CP) or PBS (control, CT; 100 uL). One to 5 weeks after the last injection, mice were sacrificed, and testes were removed, weighed, and fixed in Bouin’s solution for histological evaluation. Changes in the testes weight following CP treatment (CP) compared to control (Control) is presented ( A ). The histology of the seminiferous tubules was examined by hematoxylin-eosin staining ( B ) and a summary of seminiferous tubule damage after 1–5 weeks post CP (CP) treatment compared to the CT is presented ( C ). Ten days post-treatment, the histology of the seminiferous tubules was evaluated by H&E staining ( D ), testes were weighed ( E ), and the total number of cells isolated from the seminiferous tubules were counted ( F ). The presence of VASA-, GFR-α-1-, α-6-Integrin-, CD9-, and C-KIT-positive stained cells in the seminiferous tubules of CT and CP-treated immature mice ( G – K ) was examined by immunofluorescence staining (IF) using specific primary antibodies and Cy3 or Alexa-flour 488 with the relevant secondary antibodies (VASA, α-6-Integrin, CD9, and C-KIT red staining and GFR-α-1 green staining). DAPI (blue color) stained the nucleus of the cells. Arrows show the location of stained cells in the testicular tissues. As a negative control (NC), we stained the tissues only with the secondary antibodies (NC for α-6-Integrin, CD9 and C-KIT were similar and therefore, we present only NC for α-6-Integrin). ( B )—X20 light microscope magnification (100 µm scale). ( D )—X40 light microscope magnification (100 µm scale). ( G – K )—X40 fluorescent microscope magnification (100 µm scale). **— p < 0.01 and ***— p < 0.001.

Article Snippet: Following the removal of the blocking buffer, the first antibodies were added, as follows: Monoclonal mouse anti-mouse Vimentin (Novus, Littleton, CO, USA; 1:500), and polyclonal goat anti-mouse α- sma (Abcam, 1:250), Polyclonal goat anti-mouse Integrin α6 (Santa Cruz, CA, USA; 1:40), polyclonal rabbit anti-mouse VASA (Santa Cruz; 1:100), polyclonal rabbit anti-mouse CD9 (Santa Cruz; 1:100), monoclonal mouse anti-mouse GFR-α-1 (Santa Cruz, sc-271546; 1:50), monoclonal mouse anti-mouse α-6-INTEGRIN (Santa Cruz, 1:50), monoclonal mouse anti-mouse CD9 (Santa Cruz, 1:50), and monoclonal mouse anti-mouse C-KIT (Santa Cruz, 1:50), polyclonal rabbit anti-mouse BOULE (Santa Cruz; 1:50), polyclonal rabbit anti-mouse CREM-1 (Santa Cruz; 1:50), and polyclonal rabbit anti-mouse ACROSIN (Santa Cruz; 1:200).

Techniques: Injection, Control, Staining, Isolation, Immunofluorescence, Negative Control, Light Microscopy, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

doi: 10.3390/ijms22041672

Figure Lengend Snippet: CP-treated immature mice showed a significant decrease in the number of subpopulations of spermatogenic cells compared to control: Cyclophosphamide (CP)- or PBS-treated mice (control, CT) were i.p injected as described in . Ten days post-treatment, testes were removed, seminiferous tubules were separated, and cells were enzymatically isolated from the seminiferous tubules. The premeiotic cells that express α-6-INTEGRIN, VASA, CD9, GFR-α, and c-KIT, or the meiotic cells that express the markers BOULE and CREM and the meiotic/post-meiotic cells that express the marker ACROSIN were identified by immunofluorescence staining using specific primary antibodies for each cell marker and the secondary antibody Cy3 (red color). DAPI (blue color) stained the nucleus of the cells (( A , B ), respectively). The identified premeiotic, meiotic, and meiotic/post-meiotic cells were counted, and their number/testis was evaluated (( C , D ), respectively). Arrows indicate the stained cells. ***— p < 0.001.

Article Snippet: Following the removal of the blocking buffer, the first antibodies were added, as follows: Monoclonal mouse anti-mouse Vimentin (Novus, Littleton, CO, USA; 1:500), and polyclonal goat anti-mouse α- sma (Abcam, 1:250), Polyclonal goat anti-mouse Integrin α6 (Santa Cruz, CA, USA; 1:40), polyclonal rabbit anti-mouse VASA (Santa Cruz; 1:100), polyclonal rabbit anti-mouse CD9 (Santa Cruz; 1:100), monoclonal mouse anti-mouse GFR-α-1 (Santa Cruz, sc-271546; 1:50), monoclonal mouse anti-mouse α-6-INTEGRIN (Santa Cruz, 1:50), monoclonal mouse anti-mouse CD9 (Santa Cruz, 1:50), and monoclonal mouse anti-mouse C-KIT (Santa Cruz, 1:50), polyclonal rabbit anti-mouse BOULE (Santa Cruz; 1:50), polyclonal rabbit anti-mouse CREM-1 (Santa Cruz; 1:50), and polyclonal rabbit anti-mouse ACROSIN (Santa Cruz; 1:200).

Techniques: Control, Injection, Isolation, Marker, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

doi: 10.3390/ijms22041672

Figure Lengend Snippet: Effect of hormones (FSH and testosterone) and cytokines (IL-1α and TNFα) on the proliferation and differentiation of spermatogonial cells isolated from CP-treated immature mice cultured in vitro in MCS.

Article Snippet: Following the removal of the blocking buffer, the first antibodies were added, as follows: Monoclonal mouse anti-mouse Vimentin (Novus, Littleton, CO, USA; 1:500), and polyclonal goat anti-mouse α- sma (Abcam, 1:250), Polyclonal goat anti-mouse Integrin α6 (Santa Cruz, CA, USA; 1:40), polyclonal rabbit anti-mouse VASA (Santa Cruz; 1:100), polyclonal rabbit anti-mouse CD9 (Santa Cruz; 1:100), monoclonal mouse anti-mouse GFR-α-1 (Santa Cruz, sc-271546; 1:50), monoclonal mouse anti-mouse α-6-INTEGRIN (Santa Cruz, 1:50), monoclonal mouse anti-mouse CD9 (Santa Cruz, 1:50), and monoclonal mouse anti-mouse C-KIT (Santa Cruz, 1:50), polyclonal rabbit anti-mouse BOULE (Santa Cruz; 1:50), polyclonal rabbit anti-mouse CREM-1 (Santa Cruz; 1:50), and polyclonal rabbit anti-mouse ACROSIN (Santa Cruz; 1:200).

Techniques: Isolation, Cell Culture, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

doi: 10.3390/ijms22041672

Figure Lengend Snippet: Isolated cells from seminiferous tubules of CP-treated immature mice developed colonies in vitro in methylcellulose culture system (MCS): Isolated cells from seminiferous tubules of CP-treated immature mice, ten days after the last injection were cultured in a methylcellulose culture system (MCS). The MCS was composed of 42% methylcellulose, KSR (10%), StemPro, and growth factors (GDNF, LIF, FGF, EGF) as described in materials and methods section in the absence or presence of IL-1α, TNF-α, FSH, testosterone (T), or both IL-1α + T, TNF-α + T, FSH + T. Developed colonies after 4–5 weeks of culture are presented ( A ). The developed cells in the different treatments were positively stained for premeiotic markers (VASA, CD9, α-6-integrin, C-KIT), meiotic markers (Boule, Crem) and meiotic/post-meiotic marker (Acrosin) as examined by immunofluorescence staining using specific primary antibodies for each cell type and the secondary antibody Cy3 (red color) and DAPI (blue color) that stained the nucleus of the cells ( B ). Scale bare: 100 μm.

Article Snippet: Following the removal of the blocking buffer, the first antibodies were added, as follows: Monoclonal mouse anti-mouse Vimentin (Novus, Littleton, CO, USA; 1:500), and polyclonal goat anti-mouse α- sma (Abcam, 1:250), Polyclonal goat anti-mouse Integrin α6 (Santa Cruz, CA, USA; 1:40), polyclonal rabbit anti-mouse VASA (Santa Cruz; 1:100), polyclonal rabbit anti-mouse CD9 (Santa Cruz; 1:100), monoclonal mouse anti-mouse GFR-α-1 (Santa Cruz, sc-271546; 1:50), monoclonal mouse anti-mouse α-6-INTEGRIN (Santa Cruz, 1:50), monoclonal mouse anti-mouse CD9 (Santa Cruz, 1:50), and monoclonal mouse anti-mouse C-KIT (Santa Cruz, 1:50), polyclonal rabbit anti-mouse BOULE (Santa Cruz; 1:50), polyclonal rabbit anti-mouse CREM-1 (Santa Cruz; 1:50), and polyclonal rabbit anti-mouse ACROSIN (Santa Cruz; 1:200).

Techniques: Isolation, In Vitro, Injection, Cell Culture, Staining, Marker, Immunofluorescence

Journal: Journal of Nanobiotechnology

Article Title: Human trophoblast-derived exosomes attenuate doxorubicin-induced cardiac injury by regulating miR-200b and downstream Zeb1

doi: 10.1186/s12951-020-00733-z

Figure Lengend Snippet: Characterization of TSC-derived exosomes. a The ultrastructure of exosomes was analyzed by transmission electron microscopy. b Western blot analysis showed the expression of protein markers of exosomes, such as CD63, CD9 and TSG101. c Representative DLS number distribution measurement of the isolated exosome population demonstrated an average diameter of 101 nm

Article Snippet: Then, the membranes were incubated with primary rabbit anti-mouse antibodies against CD9 (Cat: ab92726), CD63 (Cat: ab213090), TSG101 (Cat: ab125011), cleaved-caspase3 (Cat: ab2302), Bcl-2 (Cat: ab32124), Zeb1 (Cat: ab81972), p65 (Cat: ab16502) and GAPDH (Cat: ab181602) (Abcam; USA) at a dilution of 1:1000.

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Isolation