Sample collection was done as previously described [8 (link)]. Briefly, twenty-four hours after infection, challenged mice were isoflurane anesthetized for retro-orbital blood collection and euthanized by cervical dislocation. Mesenteric adipose tissue (MAT, the visceral adipose tissue between the two peritoneal layers of the mesentery) was removed, its weight was measured, and the tissue was placed in Hanks’s balanced salt solution supplemented with 4% bovine serum albumin (BSA) and 10 mM HEPES buffer (all from Sigma-Aldrich) for further analysis. To minimize variability, all removed mesenteric adipose tissue was used for isolation of adipocyte fraction and stromal vascular fraction cells. Subcutaneous adipose tissue (SAT) and gonadal adipose tissue (GAT) were also removed, their weight was measured, and the latter was preserved in formaldehyde 3.7–4.0% buffered to pH = 7 (Panreac, Darmstadt, Germany) for immunohistochemical analysis. The liver was also collected, some portions were preserved in formaldehyde 3.7–4.0% buffered to pH = 7 (Panreac, Darmstadt, Germany) for immunohistochemical analysis and others were stored in TRI Reagent® (Sigma) for RNA extraction.
Hanks s balanced salt solution
Manufactured by Merck Group
Hanks's balanced salt solution is a widely used isotonic buffer solution that maintains the pH and osmotic pressure of cell cultures. It contains a balanced mixture of inorganic salts, including sodium, potassium, calcium, and magnesium, as well as glucose and phenol red. This solution is commonly used to support the viability and growth of cells in various in vitro applications.
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2 protocols using hanks s balanced salt solution
1
Adipose Tissue and Liver Analysis
2
Tumor Inoculation and Characterization
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CT26 (0.2 × 106), B16 (0.5 × 106) or Pan02 (1 × 106) tumor cells were suspended in 200 µL phosphate buffered saline, and injected subcutaneously into the right flank of recipient mice. For orthotopic modeling, we inoculated 0.1 × 106 4T1 cells into the fat pad of the abdominal mammary gland. Mice were randomized to different treatment arms, with tumor sizes distributed evenly between the treatment groups. Tumor size was measured with a Vernier caliper, and tumor volume was calculated based on the perpendicular diameters of individual tumors ( ). Only mice that reached the endpoint, defined as a maximal tumor volume of 1500 mm3, were included in the survival analysis.
For flow cytometry and gene expression analysis, tumors were harvested 6 days after the last treatment dose. The harvested tissue was dissociated through incubation for 45 min at 37°C and 650 rpm in Hanks’s Balanced salt solution (Sigma Aldrich, Cat: 55021C) containing 75 μg/mL DNase (Sigma Aldrich, Cat: DN25) and 2 mg/mL of collagenase IV (Millipore, Cat: C5138).
For flow cytometry and gene expression analysis, tumors were harvested 6 days after the last treatment dose. The harvested tissue was dissociated through incubation for 45 min at 37°C and 650 rpm in Hanks’s Balanced salt solution (Sigma Aldrich, Cat: 55021C) containing 75 μg/mL DNase (Sigma Aldrich, Cat: DN25) and 2 mg/mL of collagenase IV (Millipore, Cat: C5138).
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