The cord blood derived CD34+ HPCs were plated on fibronectin-coated tissue culture flasks and cultured in endothelial basal medium (EBM-2, Lonza) supplemented with EGM-2-MV-SingleQuots (Lonza) and 1% penicillin-streptomycin (Sigma-Aldrich) in a humidified incubator at 37°C with 5% CO2. After 4 days of culture, nonadherent cells were discarded by washing with PBS. Medium were changed every 2 days thereafter. To confirm the EPC phenotype after culturing for 7 days, adherent cells were incubated with DiI-labeled Ac-LDL (Molecular Probes) for one hour before visualizing with an inverted fluorescent microscope (Leica DM IRE2). Immunostaining with antibodies against VEGFR2(Flk-1,1∶50 dilution; Santa Cruz Biotechnology), CD31(1∶100 dilution; Cell Signaling) followed by Cy3-conjugated goat anti-mouse antibody(1∶250 dilution; Invitrogen) was performed, and then cells were visualized with an inverted fluorescent microscope (Leica DM IRE2). After culturing for 14 days, cells were stained with anti-von Willebrand factor antibody FITC (1∶100 dilution; Abcam), or stained with antibodies against VEGFR2 (Flk-1,1∶50 dilution; Santa Cruz Biotechnology), CD31(1∶100 dilution; Cell Signaling) followed by Cy3-conjugated goat anti-mouse antibody(1∶250 dilution; Invitrogen), and then visualized with an inverted fluorescent microscope(Leica DM IRE2).
Cy3 conjugated goat anti mouse antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Cy3-conjugated goat anti-mouse antibody is a secondary antibody used in various immunological techniques. It is designed to bind to mouse primary antibodies, allowing for the detection and visualization of target proteins or antigens in a sample. The Cy3 fluorescent dye conjugated to the antibody emits light in the red-orange spectrum, enabling the labeled targets to be detected using appropriate imaging equipment and techniques.
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Lab products found in correlation
Penicillin streptomycin, by Merck Group (1 mentions)
Egm 2 mv singlequots, by Lonza (1 mentions)
Ebm 2, by Lonza (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ea 53, by Merck Group (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Axio imager z2 microscope, by Zeiss (1 mentions)
Bz x710 microscope, by Keyence (1 mentions)
Metafer4, by MetaSystems (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Mms 120r, by Fortrea (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Fitc conjugated goat anti mouse antibody, by Abcam (1 mentions)
γh2ax antibody, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
β catenin antibody, by Proteintech (1 mentions)
6 protocols using cy3 conjugated goat anti mouse antibody
1
Endothelial Progenitor Cell Characterization
2
Visualization of EMP Uptake by HUVECs
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As previously described,24 (link) micro-wells (µ-Slides, Ibidi) were coated overnight with 50 µg/mL recombinant annexin-V. Purified EMPs, diluted in NaCl/Hepes buffer (with CaCl2, 10mM), were incubated at 37 °C for 30 minutes in the presence of CFSE (0.2 mM). Then, CFSE-labeled EMPs were then seeded in coated micro-wells and were immobilized in the dark at room temperature. When appropriate, experiments were also performed to record the change in the number of EMPs after fixation for 1 h.
ECs uptake experiments were performed according to the following method. In brief, pelleted EMPs diluted in PBS were incubated with 0.2 mM CFSE at 37 °C for 30 minutes, washed and centrifuged twice at 20,000 ×g, and resuspended in sterile PBS. HUVECs were incubated with CFSE-labeled EMPs for different time frames. After three washing steps, HUVECs were fixed in 4% paraformaldehyde (PFA), blocked with 5% bovine serum albumin (BSA) and stained with anti-CD31 (mouse monoclonal antibody, 1:10, Thermo Fisher Scientific, USA, cat.no. MA513188) followed by incubation with Cy3-conjugated goat anti-mouse antibody (1:500, Thermo Fisher Scientific, USA, cat.no. A10521). After washing three times, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:2000, Thermo Fisher Scientific, USA, cat.no. D3571). The spinning disc confocal microscopy was used to visualize the cellular uptake of EMPs.
ECs uptake experiments were performed according to the following method. In brief, pelleted EMPs diluted in PBS were incubated with 0.2 mM CFSE at 37 °C for 30 minutes, washed and centrifuged twice at 20,000 ×g, and resuspended in sterile PBS. HUVECs were incubated with CFSE-labeled EMPs for different time frames. After three washing steps, HUVECs were fixed in 4% paraformaldehyde (PFA), blocked with 5% bovine serum albumin (BSA) and stained with anti-CD31 (mouse monoclonal antibody, 1:10, Thermo Fisher Scientific, USA, cat.no. MA513188) followed by incubation with Cy3-conjugated goat anti-mouse antibody (1:500, Thermo Fisher Scientific, USA, cat.no. A10521). After washing three times, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:2000, Thermo Fisher Scientific, USA, cat.no. D3571). The spinning disc confocal microscopy was used to visualize the cellular uptake of EMPs.
3
Quantifying Cardiomyocyte Size via Immunofluorescence
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For immunofluorescence-based quantification of NRVM size, cells were fixed with 4% paraformaldehyde in DPBS and permeabilized by 0.25% Triton X-100. Cells were incubated with mouse monoclonal antibody against sarcomeric α-actinin at 3 µg/mL (EA-53; Sigma-Aldrich, Taufkirchen, Germany) in 2.5% BSA in DPBS for 1 h followed by secondary Cy3-conjugated goat anti-mouse antibody at 2 µg/mL (Thermo Fisher Scientific, Bremen, Germany) in 2.5% BSA in DPBS for 30 min. For visualization of nuclei, NRVM were incubated with 4’,6-diamidino-2-phenylindole at 200 ng/mL (DAPI; Sigma-Aldrich, Taufkirchen, Germany) in DPBS for 12 min. Images of NRVM were taken on a Zeiss Axio Observer Z1 microscope (Carl Zeiss Microscopy, Goettingen, Germany) with a 20× objective. Average cardiomyocyte cell size was quantified by measuring at least 100 cells per group using Carl Zeiss Zen software.
4
Immunofluorescence Staining Protocol for Phospho-Histone H2AX
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Immunofluorescence staining was performed as described previously (18 (link)). Briefly, cells were fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature, and then permeabilized with 0.5% Triton X-100 in PBS for 10 minutes at room temperature. The cells were incubated for 30 minutes at 37°C with a mouse monoclonal anti-phospho-histone H2AX antibody (1:40,000; Upstate), in PBS containing 1% bovine serum albumin (BSA). The secondary antibody was Cy3-conjugated goat anti-mouse antibody (1:1000; Invitrogen). Cells were mounted using Vectashield containing DAPI (Vector Laboratories) and observed with a BZ-X710 microscope (Keyence) or an Axio Imager Z2 microscope (Carl Zeiss) equipped with the Metafer4 software (MetaSystems).
5
Immunofluorescence Imaging of Nuclear Pore Complexes
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Xenopus S3 cells were grown in 70% Leibovitz L-15 media GlutaMAX Supplement, 10% FBS and 500 units/ml penicillin-streptomycin (all from Gibco). Cells quickly washed with 70% PBS supplemented with 0.1 (v/v)% Triton X-100 and fixed for 5 min in 2% paraformaldehyde supplemented with 0.5 (v/v)% Triton X-100. Immunofluorescence analysis was performed as described17 (link). The rabbit polyclonal antibody was used in a 1:100 dilution and visualized by an Alexa 488-conjugated goat anti-rabbit antibody (Invitrogen) diluted 1:2,000. Mab414 ascites (Covance, MMS-120R), which marks nuclear pore complexes, was used in a 1:2,000 dilution and visualized by a Cy3-conjugated goat anti-mouse antibody (Invitrogen) diluted 1:2,000. Chromatin was stained with 10 μg/ml DAPI. Fluorescence images were acquired using a confocal microscope (LSM780, Zeiss) using 405-, 488-, and 561-nm laser lines and an apochromat 63 × NA 1.40 oil DIC M27 objective.
6
Immunofluorescence Staining Protocol
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The slides were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 (Beyotime Biotechnology) for 30 min. After blocking in 1% BSA for 15 min at room temperature, cells were incubated with primary antibody overnight at 4 °C. The primary antibodies were as follows: γH2AX antibody (1:200 diluted, Thermo Fisher Scientific), USP43 antibody (1:50 diluted, Santa Cruz Biotechnology), HDAC2 antibody (1:100 diluted, Proteintech Group) and β-catenin antibody (1:50 diluted, Proteintech Group). Then cells were incubated with Cy3-conjugated goat anti-rabbit antibody (1:200 diluted, Invitrogen), Cy3-conjugated goat anti-mouse antibody (1:200 diluted, Invitrogen) or FITC-conjugated goat anti-mouse antibody (1:200 diluted, Abcam) for 60 min at room temperature in the dark. After washing with PBS, cells were counterstained with DAPI (Aladdin) to mark the nuclei. The slides were sealed with fluorescent mounting medium (Solarbio Science), and observed under the microscope.
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