YAC128 HD mice and WT littermates were obtained from The Jackson Laboratory (Bar Harbor, ME, USA; JAX Mice #004938, FVBN/J background). Additionally, for the assessment of fibroblast reprogramming, we generated a crossbreed of YAC128 and Oct-eGFP mice (Lengner et al., 2007 (link)) (JAX Mice #008214, B6;129S4/SvJae background). The cultured mouse adult fibroblasts were derived from back-skin biopsies of 8- to 10-week-old mice. Briefly, the skin fibroblasts were cultured using the explant technique in HEPES-buffered DMEM (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% FBS originating from New Zealand (Sigma-Aldrich), 2 mM L-Gln (Sigma-Aldrich), 1× antibiotic-antimycotic mixture (Sigma-Aldrich) and 10 μg/ml L-ascorbic acid (Sigma-Aldrich). Human HD fibroblasts, line GM04281, were obtained from Coriell (Coriell Cell Repository, Camden, NJ, USA). The protocols for the maintenance and handling of the animals were approved and monitored by the Local Ethical Commission for Animal Experiments in Poznan.
Hepes buffered dmem
Manufactured by Merck Group
Sourced in United States
HEPES-buffered DMEM is a cell culture medium that contains the buffer HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) instead of the standard sodium bicarbonate buffer. The HEPES buffer helps maintain a stable pH in the medium, which is important for cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
L gln, by Merck Group (1 mentions)
Antibiotic antimycotic mixture, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Jax mice, by Jackson ImmunoResearch (1 mentions)
Liberase, by Roche (1 mentions)
Moflo cell sorter, by Beckman Coulter (1 mentions)
2 protocols using hepes buffered dmem
1
Generation and Characterization of HD Mouse Models
2
Isolation of Adipose Tissue Macrophages
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To isolate ATMs from lean and obese adipose, male C57BL/6J mice were randomized onto either chow or an obesogenic diet (HFD, 45% kcal from fat, HFD; D06011802, Research Diets, New Brunswick, NJ) at weaning. After 23 weeks on diet, epididymal white adipose tissue (eWAT) was minced in 25 mM HEPES-buffered DMEM/1% fatty acid-free, low endotoxin BSA (Sigma) and completely digested with 0.5 mg/mL Liberase TM (Roche Diagnostics, Indianapolis, IN) at 37°. Samples were centrifuged at 200× g for 10 min at 4° to pellet the MΦ-rich stroma vascular fraction (SVF). The pellet was washed twice with in 25 mM HEPES-buffered DMEM/1% fatty acid-free, low endotoxin BSA and red blood cells were lysed and washed again. Samples were stained with anti-F4/80-PE and sorted on a MoFlo cell sorter (Beckman Coulter, Brea, CA). F4/80hi MΦs were collected for mRNA isolation and gene expression analysis.
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