Dnaase

At the time of killing, most of the adrenal glands were collected fresh for preparation of a purified, isolated glomerulosa cell preparation as previously reported by us (Braley et al. 1981 (link), 1996 (link), Baudrand et al. 2015 (link), Garza et al. 2015 (link), Chong et al. 2017 (link)). In brief, adrenals were bisected and by blunt scraping the zona glomerulosa (ZG) capsular layer was separated from the fasciculata/medulla. The capsules were suspended in Krebs Ringer bicarbonate solution (Sigma-Aldrich) (0.1% BSA, 200 mg glucose/dl, L-glutamine, 3.7 mmol/L of K⁺) (KRBGA) solution with collagenase (3.7 mg/mL) and DNAase (0.05 mg/mL) (Worthington Biochemical, Freehold, NJ, USA) for 60-min incubation at 37°C under 95% O₂ and 5% CO₂. Isolated ZG cells underwent three rounds of brief washing and centrifugation followed by determination of cell count. Purity of the preparation was determined as previously described (Braley et al. 1981 (link), 1996 (link)).
Equal amounts of cells (~50,000) were incubated with 10⁻⁷ M angiotensin II (ANGII) or 8.7 mM potassium (K⁺) for 60 min at 37°C under 95% O₂ and 5% CO₂. Basal as well as ANGII and K⁺-stimulated aldosterone secretion levels were determined, and the aldosterone levels normalized to 1 million cells.

Immediately after sacrifice, mice were perfused with 10ml PBS to eliminate blood leukocyte contamination from the lungs. Lungs from each mouse were excised, washed in RPMI, minced, enzymatically digested in media [RPMI with 25mM HEPES, 5% FBS, GlutaMAX, penicillin, streptomycin, non-essential amino acids, and βMe (Life Technologies)] with collagenase (1mg/ml, Roche, Indianapolis, IN) and 500 U/ml DNAase (Worthington) at 37° C for 30 min, and dispersed using a GentleMACs tissue dissociator. Erythrocytes were lysed ammonium chloride buffer for 3 minutes followed by neutralization with excess of RPMI. The resultant single cells suspension was filtered through sterile 100 um nylon mesh (Nitex, Kansas City, MO) and leukocytes were purified using a 20% Percoll (GE) gradient. Spleens were dispersed through a 70uM cell strainer using a syringe plunger and erythrocytes were lysed with ammonium chloride. Isolated cells were suspended in RPMI with 25mM HEPES supplemented with 10% FBS, GlutaMAX, penicillin, streptomycin, non-essential amino acids, and βMe (Life Technologies) and counted on a hemocytometer prior to use in all experiments. Differential cell counts were performed on Diff-Quik stained cytospin samples. Total numbers were calculated by multiplying the percentage of each cell type by the total number of leukocytes per lung.