Spleen and bone marrow were prepared as described previously (Yu et al., 2013 (link)). The peripheral blood monocytes (PBMCs) were isolated using Histoplaque (Sigma). Wound sites were minced and digested with 1.0 mg/ml collagenase (Sigma) to generate a single cell suspension and purified using a Percoll gradient (Sigma). These dissociated single cells were stained with fluorochrome-conjugated antibodies specific for mouse CD11b, Ly6G, CD45 (eBioscience) and CCR2 (R&D). For COL1A1 intracellular staining, cells were harvested, stained with surface markers, fixed with a fixation solution (eBioscience) for 15 min, permeabilized with ice-cold pure methanol for 30 min and incubated with biotin-conjugated mouse COL1A1 antibody (Rockland) and fluorochrome-conjugated streptavidin (eBioscience). Flow cytometry was performed using FACS Aria II (BD) and data analyzed with Flowjo.
Histoplaque
Manufactured by Merck Group
Histoplaque is a laboratory equipment product manufactured by Merck Group. It is used for the detection and identification of histoplasma capsulatum, a fungus that can cause the infectious disease histoplasmosis. The Histoplaque product provides a reliable method for the analysis and diagnosis of this condition.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase, by Merck Group (2 mentions)
Biotin conjugated mouse col1a1 antibody, by Rockland Immunochemicals (2 mentions)
Fluorochrome conjugated streptavidin, by Thermo Fisher Scientific (2 mentions)
Percoll gradient, by Merck Group (2 mentions)
Facsaria 2, by BD (2 mentions)
Collagenase 5, by Roche (1 mentions)
3 protocols using histoplaque
1
Isolation and Characterization of Wound-Associated Immune Cells
2
Isolation and Culture of Mouse Islets
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Islets were isolated from 8 to 12 weeks old C57BL/6 male mice as described before by our laboratory [29] (link). Briefly, mouse islets were isolated using perfusion and digestion of pancreas with collagenase V (from Roche), density gradient purification with histoplaque (Sigma), and then hand-picked. Isolated islets were cultured overnight in RPMI 1640 containing 10% FBS, 11 mM glucose, and then switched to RPMI medium containing 3.3 mM glucose for 1 hr before treatment with high concentration of glucose. Insulin levels in the culture media were measured with an ELISA kit from ALPCO. For Ets1 induction of TXNIP studies, 200 isolated mouse islets were also cultured 16 hrs in RPMI 1640 containing 10% FBS and 11 mM glucose and switched to RPMI 1640 with 10% FBS and 20 mM glucose. Ad-EGFP or Ad-Ets1 was then added to islets in culture for 24 hrs at MOI of 100. The islets were collected for Western blot analysis of TXNIP and Ets1.
3
Isolation and Characterization of Wound-Associated Immune Cells
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