Aortic macrophages were isolated from Ldlr−/− mice fed a Western diet for 16 weeks. Mice were injected intraperitoneally (i.p.) with PBS or IFNγ (25 mg/kg = 100 U/kg) (Whitman et al., 2000 (link)) 48 hr and 24 hr prior to euthanasia. Anesthetized mice were perfused with cold PBS with 20 U/mL heparin, and the upper vasculature was isolated and dissected of adipose tissue and paraaortic lymph nodes. The intima in the arch and innominate artery were excised and incubated in digestion media containing collagenase, hyaluronidase, and DNase1 in Hank’s balanced salt solution (HBSS) for 1 hr (Butcher et al., 2011 ). Macrophages were isolated using anti-CD11b microbeads (Miltenyl Biotek) and plated in a 96-well dish in serum-free DMEM for 24 hr. Macrophage purity was assessed by flow cytometry on a Cantos II (BD Biosciences) and analyzed by FlowJo software (v.10.4.1).
Cantos 2
Manufactured by BD
The Cantos II is a versatile laboratory equipment designed for a range of applications. It features a compact and durable construction. The core function of the Cantos II is to perform fundamental laboratory tasks.
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2 protocols using cantos 2
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Isolation and Characterization of Aortic Macrophages
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Isolation and Characterization of Aortic Macrophages
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Aortic macrophages were isolated from Ldlr−/− mice fed a Western diet for 16 weeks. Mice were injected intraperitoneally (i.p.) with PBS or IFNγ (25 mg/kg = 100 U/kg) (Whitman et al., 2000 (link)) 48 hr and 24 hr prior to euthanasia. Anesthetized mice were perfused with cold PBS with 20 U/mL heparin, and the upper vasculature was isolated and dissected of adipose tissue and paraaortic lymph nodes. The intima in the arch and innominate artery were excised and incubated in digestion media containing collagenase, hyaluronidase, and DNase1 in Hank’s balanced salt solution (HBSS) for 1 hr (Butcher et al., 2011 ). Macrophages were isolated using anti-CD11b microbeads (Miltenyl Biotek) and plated in a 96-well dish in serum-free DMEM for 24 hr. Macrophage purity was assessed by flow cytometry on a Cantos II (BD Biosciences) and analyzed by FlowJo software (v.10.4.1).
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