The murine microglial cell-line (BV-2) was obtained from Dr. R. Donato (University of Perugia, Perugia, Italy) (Blasi et al, 1990 (link)). The BV-2 cultures used in our experiments were 100 % positive for the microglia marker, mouse CD68 (macrosialin), as determined by flow cytometric analyses using Alexa Fluor® 647-conjugated anti-CD68 antibody (0.025 µg per 2.5×105 cells in 100 µL volume; BioLegend Inc., San Diego, CA, USA; #137003). Human embryonic kidney 293 T (HEK 293 T) cells were obtained from Invitrogen/Life Technologies (Grand Island, NY, USA). BV-2 cells and HEK 293 T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10 % fetal bovine serum (FBS), 2 mM glutamine, and antibiotics, by standard procedures. Human microglial cells that were isolated from fetal human brain were obtained from Clonexpress (Gaithersburg, MD, USA) and were maintained in 50:50 DMEM:F-12 supplemented with 5 % FBS and 10 ng/mL of M-CSF.
Alexa fluor 647 conjugated anti cd68 antibody
Manufactured by BioLegend
Sourced in United States
Alexa Fluor® 647-conjugated anti-CD68 antibody is a fluorescently-labeled monoclonal antibody that binds to the CD68 protein, a glycoprotein expressed on the surface of macrophages and monocytes. This antibody is conjugated to the Alexa Fluor® 647 fluorescent dye, which can be detected using flow cytometry or fluorescence microscopy.
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2 protocols using alexa fluor 647 conjugated anti cd68 antibody
1
Murine and Human Microglial Cell Cultures
2
BrdU Labeling and Aortic Endothelial Analysis
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For BrdU labeling experiments, mice received a single 0.2 mL i.v. injection of 2 mg BrdU in PBS, as described51 (link). Aortas were harvested after 3 or 24 h. En face immunostaining was performed as previously described51 (link). Mice were perfused with PBS, followed by 4% paraformaldehyde (PFA). The aortic arch was harvested, the periadventitial fat was removed and the aorta was fixed for 1 h in 4% PFA. After permeabilization with 0.5% Triton X-100 and 10% DMSO for 5 min at room temperature (RT), the aortic arch was incubated with Alexa Fluor® 647-conjugated anti-CD68 antibody (BioLegend) or Alexa Fluor® 647-conjugated anti-CD45 (BioLegend) and biotin-conjugated anti-BrdU antibodies overnight at 4 °C, followed by CyTM3-Streptavidin. Nuclei were stained with Hoechst 33342. The aorta was opened and mounted on a glass slide. En face immunofluorescence confocal images were obtained using Nikon A1R confocal microscope (Nikon, Tokyo, Japan) equipped with ×40 oil objectives. Every 2 mm z-stack slices were aligned and compressed to create a maximum intensity projection image. ImageJ software was used for analysis.
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