Adipose tissue macrophages were isolated as previously described.13 (link) Briefly, whole adipose tissue samples were cut into small pieces, gently homogenized in RPMI using disposable tissue grinders (Fisher Brand), and filtered through 70μm cell strainers (BD Flacon, Bedford, MA) to obtain single cell suspension. Macrophages were isolated by dual density gradient (Histopaque -1077 and Histopaque-1119, Sigma Aldrich). Isolated cells were incubated with FACS permeabilization solution 2 (BD Biosciences) for 30 min at 37 C, then measured by flow cytometry for cells positive for both CD14 (BD Pharmingen), and hVEGF-A total (which does not distinguish isoforms) (R&D systems).
Facs permeabilization solution 2
Manufactured by BD
FACS permeabilization solution 2 is a buffer solution designed for use in flow cytometry applications. It facilitates the permeabilization of cell membranes, allowing for the intracellular staining and analysis of cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Disposable tissue grinders, by Thermo Fisher Scientific (1 mentions)
Histopaque 1119, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Live dead fixable near ir dead cell stain, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Facsverse, by BD (1 mentions)
2 protocols using facs permeabilization solution 2
1
Isolation of Adipose Tissue Macrophages
2
Quantification of Cytokine-Producing T Cells
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Tracheobronchial LN cells were restimulated as above, in duplicates of 5×106 cells/calf for 18 hours. Brefeldin A (Sigma) was added for the last 15 hours at 10 µ g/ml. Cells were stained for viability (LIVE/DEAD Fixable Near-IR Dead Cell Stain, Life Technologies) and for expression of surface markers, CD4 and CD8 (MCA1653F:FITC (CD4), MCA837A647: AlexaFlour 647 (CD8), AbD Serotec). Cells were then fixed for 10
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