Mouse brain tissues were digested, followed by neutralization and pipetting, and then passed through a 30-μm cell strainer. Myelin was removed by 70% Percoll centrifugation. Derived single-cell suspensions were either analyzed using LSR II Flow Cytometer (BD Biosciences) or purification of microglia and astrocytes with magnetic beads. For FACS analysis of microglia, cells were first blocked with anti-mouse CD16/CD32 (BioLegend) prior to staining with antibodies against surface or intracellular markers. To purify microglia and astrocytes, cell suspensions were also blocked with anti-mouse CD16/CD32, followed by anti-Cx3cr1-PE (BioLegend) and anti-ACSA-2-PE antibodies (Miltenyi), respectively, along with anti-PE MicroBeads (Miltenyi) to capture bound cells. For the in vivo phagocytosis assay, 0.5 μL of FITC-labeled zymosan particle suspension was infused into the hippocampus via cannula. Microglia were purified by the Percoll centrifugation, stained, and analyzed by FACS analysis (Supplemental Experimental Procedures).
Anti acsa 2 pe
Manufactured by Miltenyi Biotec
Sourced in United States
Anti-ACSA-2-PE is a laboratory reagent that can be used to detect the presence of ACSA-2 (Adenylyl Cyclase-Associated Protein 2) in samples. It is conjugated with the fluorescent dye Phycoerythrin (PE) to enable visualization and quantification of ACSA-2-positive cells or molecules.
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Lab products found in correlation
3 protocols using anti acsa 2 pe
1
Isolation and Analysis of Microglia and Astrocytes from Mouse Brain
2
Isolation and Analysis of Microglia and Astrocytes from Mouse Brain
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Mouse brain tissues were digested, followed by neutralization and pipetting, and then passed through a 30-μm cell strainer. Myelin was removed by 70% Percoll centrifugation. Derived single-cell suspensions were either analyzed using LSR II Flow Cytometer (BD Biosciences) or purification of microglia and astrocytes with magnetic beads. For FACS analysis of microglia, cells were first blocked with anti-mouse CD16/CD32 (BioLegend) prior to staining with antibodies against surface or intracellular markers. To purify microglia and astrocytes, cell suspensions were also blocked with anti-mouse CD16/CD32, followed by anti-Cx3cr1-PE (BioLegend) and anti-ACSA-2-PE antibodies (Miltenyi), respectively, along with anti-PE MicroBeads (Miltenyi) to capture bound cells. For the in vivo phagocytosis assay, 0.5 μL of FITC-labeled zymosan particle suspension was infused into the hippocampus via cannula. Microglia were purified by the Percoll centrifugation, stained, and analyzed by FACS analysis (Supplemental Experimental Procedures).
3
Isolation and Analysis of Astrocytes from Stressed Mice
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The hippocampus was extracted from unstressed and stressed mice. The tissue was digested by treatment with 0.25% trypsin (containing 0.02% EDTA) for 10 min, and DMEM (Invitrogen, USA) with 10% FBS was used to stop digestion. The cells and tissue debris were centrifuged for 5 min at 1000 rpm, and after suspending in PBS, the mixtures were filtered with a 200 mesh sieve. We incubated anti-ACSA-2-PE (Miltenyi Biotec, USA) and IgG2b Isotype Control-PE (Miltenyi Biotec, USA) with the cells and sorted the astrocyte population by flow cytometry (FACSAria II SORP, BD, USA). These cells were used to detect the release of inflammatory factors such as IL-1β, IL-6, and TNF-α from stressed mice.
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