EPCs were fixed in 4% PFA for 15 min at room temperature. Cells were washed, permeabilized with 0.1% Triton X-100 in PBS for 20 min and washed with PBS. Additionally, the cells were blocked with 5% normal goat serum in PBS + 0.1% Tween 20 for 1 h at room temperature. γH2A.X (Abcam) antibody was diluted with 5% BSA in PBS and incubated overnight at 4 °C. The next day, the slides were washed with PBS and incubated with the secondary antibody, Alexa Fluor-555 (Invitrogen), at room temperature for 1 h in the dark. The washed slides were mounted with DAPI. The stained cells were observed using an automated microscope (BioTek).
Automated microscope
Manufactured by Agilent Technologies
Sourced in United States
The Automated Microscope is a lab equipment product that provides automated imaging and analysis capabilities. It is designed to capture high-quality images and perform various microscopy tasks in a standardized and efficient manner.
Automatically generated - may contain errors
Lab products found in correlation
H2dcfda, by Thermo Fisher Scientific (2 mentions)
γ h2ax, by Abcam (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Accuri c6, by BD (1 mentions)
Edu cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Sa β gal staining kit, by Cell Signaling Technology (1 mentions)
Sdf 1α, by R&D Systems (1 mentions)
Heparin, by Merck Group (1 mentions)
Cd31 antibody, by Abcam (1 mentions)
Matrigel, by Corning (1 mentions)
Balb c nude mice, by Orient Bio (1 mentions)
6 protocols using automated microscope
1
Immunofluorescent Detection of γH2A.X
2
ROS Measurement Using H2DCFDA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure ROS levels, cell-permeant, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Thermo Fisher Scientific), was used. The detached cells were incubated with 1 μM H2DCFDA for 30 min at 37 °C. Subsequently, the cells were washed with PBS and analyzed using flow cytometry (Accuri C6; BD Biosciences). The cell plates were incubated with 10 μM DCFDA at 37 °C for 30 min. Hoechst 33342 was used for nuclear staining. After washing with PBS, images were analyzed using an automated microscope (BioTek).
3
Quantifying Endothelial Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EPCs were cultured on coverslips in a 6-well plate containing 10 μM EdU for 5 h. EdU staining was conducted using the EdU Cell Proliferation Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The nuclei were stained with Hoechst-33342 (Sigma-Aldrich). An automated microscope (BioTek, Winooski, VT, USA) was used to acquire the images.
4
Senescent EPC Senescence Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Senescent EPCs were seeded at 1.5 × 105 cells on a 6-well plate. The cells were stained using a SA β-gal staining kit (Cell Signaling Technology, Denvers, MA, USA), according to the manufacturer’s instructions. After staining, the images were acquired using an automated microscope (BioTek), and the cells were counted with blue-colored staining for senescence-associated β-galactosidase activity.
5
Transwell Migration Assay for Cell Chemotaxis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transwell migration assays were performed using 24-well Transwell 8.0 μm pore polycarbonate membrane inserts (Corning, NY, USA). The upper inserts were seeded at 5 × 104 cells in EGM-2 medium and lower chamber was added with EGM-2 with 100 ng/mL SDF-1α (R&D Systems, Minneapolis, MN, USA). After incubating the plates for 6 h at 37 °C, the cells were fixed in 4% paraformaldehyde (PFA) and stained with 0.5% crystal violet in 25% methanol. The inserts were washed with distilled water until clean, and the insert membranes were mounted on glass slides. The stained cells were observed using an automated microscope (BioTek).
6
Angiogenesis Assay in Nude Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Matrigel plug assay was performed on BALB/c nude mice (Orient Bio, Seongnam, Republic of Korea). Cells (5 × 105 cells) were added to 500 μL of growth factor-reduced Matrigel (Corning) containing 50 unit/mL heparin (Sigma-Aldrich) and then, subcutaneously injected into mice. Six days later, the mice were anesthetized, and Matrigel plugs were obtained. Matrigels were fixed with 4% PFA and embedded in paraffin. For immunostaining, Matrigel sections were stained with CD31 antibody (Abcam). Sections were imaged using an automated microscope (BioTek).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!