Phenol red free media

We obtained informed consent from subjects during MSG biopsy visits in compliance with the Helsinki declaration. pSS subjects met 2016 ACR/EULAR criteria.(10 (link)) Control patients recruited at the time of the MSG biopsy had normal serology (ANA ≤ 1:160 and negative anti-SSA antibody), focus score <1 on histopathology, and did not have a diagnosis of pSS, another autoimmune disease, HIV, or Hepatitis C. Thirteen pSS and 12 control subjects were recruited (Table 1).
To isolate MSG-MSCs, MSGs were collected from the recruited subjects, minced, and digested with 3 mg/mL collagenase type IV (Worthington, Lakewood, NJ) for 1 hour at 37°C.(11 (link)) The digested tissue was washed with HBSS, filtered through a 100 μM strainer, spun, and re-suspended in complete media (α-minimal essential media (MEM, Corning, Tewksbury, MA), 20% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO), 1% L-Glutamine (Corning), and 1% Penicillin/Streptomycin (Lonza, Walkersville, MD). For estrogen treatment experiments, we used phenol-red free media (Thermo Fisher Scientific, Waltham, MA) and charcoal-stripped FBS (Sigma-Aldrich). All experiments were performed on MSG-MSCs between passage two and passage eight. CFU-F was calculated by plating 100 MSG-MSCs in six, T-75 flasks. After seven days, the number of colonies with ≥ 20 cells were counted and averaged.

Sexual dimorphisms in Protein Kinase A activity in ARVMs was determined using the Protein Kinase A activity kit (Abcam, Cambridge, United Kingdom). After isolation, ARVMs from both sexes were cultured for 45 minutes then washed with phenol-red free media (ThermoFisher, Rochester, NY) and flash frozen. ARVMs were collected in lysis buffer [20 mmol/L MOPS, 50 mmol/L β-glycerolphosphate, 50 mmol/L sodium fluoride, 1mmol/M sodium vanadate, 5 mmol/L EGTA, 2 mmol/L EDTA, 1% NP40, 1 mmol/L dithiothreitol (DTT), benzamidine, phenylmethane- sulphonylfluoride (PMSF) and 10 µg/mL leupeptin and aprotinin] and this crude enzyme lysate was used for the remainder of the assay according the manufacturer’s protocol.