Cc 5002

Confluent cells growing on a culture dish were washed in HBSS (Gibco; 14170-112) and dissociated with trypsin (Sigma). Residual trypsin was neutralized using a trypsin neutralizing buffer (Lonza; CC-5002) prior to plating cells on the TGT surfaces. Medium on the TGT surfaces was switched to Fluorobrite with or without 50 ng/ml EGF according to experimental conditions. For inhibitor studies, the cells were incubated in Fluorobrite supplemented with the indicated inhibitor for the entire incubation period.

Cultured cells were washed by dispensing and aspirating 37°C HEPES buffered saline solution (Lonza, CC-5022), and then dissociated with 0.025% Trypsin/EDTA (Lonza,CC-5012) for 10 min at 37°C. Dissociated cells were aspirated using a wide-bore pipette tip and placed in a tube containing ice-cold Trypsin Neutralization Solution (Lonza, CC5002); this was repeated to maximize cell collection. Cells were then pelleted by centrifugation, resuspended in chilled HEPES, and centrifugally pelleted once more. Using a wide-bore pipette tip, the supernatant was then removed and the cell pellet was resuspended in 100ul chilled 1X DPBS. Next, 1ml of a chilled 1:1 methanol acetone mixture was added to the cells in a dropwise manner with continuous gentle agitation. Cells were incubated on ice for 1 hour, washed in PBS++, counted, and finally resuspended in cold SSC cocktail (3× Lonza AccuGENE SSC, BMA51205 + 0.04% BSA + 1mM DTT + 0.2 U/ul RNase1 inhibitor).

The hMVECs (CC-2543, Lonza) were cultured in endothelial growth medium (EGM, Lonza), according to the manufacturer’s (Lonza) specifications. Briefly, the hMVECs were maintained at 37 °C, in a humidified incubator at 5% CO₂/ 95% air until they reached 70–80% confluency (6–8 da after seeding). Only cells at passage numbers 4–7 were used in this work. These confluent hMVECs reproduced the growth-arrested, quiescent state of normal blood vessels in vivo, expressing vascular endothelial (VE)-cadherin at cell–cell junctions. Subsequently, the medium was aspirated, the adherent hMVECs were then washed with 30 mM HEPES-BSS buffer (CC-5024, Lonza) and trypsinized by adding a volume of trypsin-EDTA solution (CC-5012, Lonza) sufficient (2.0 mL) to cover the cell surface in the T25 flask and then incubated at room temperature for 2–4 (5) min. Once they appeared rounded and began to detach, the cells were harvested by sharply rapping the culture flask. The reaction was stopped by adding (4 mL) of trypsin-neutralizing agent (CC-5002, Lonza). The detached cells were transferred to a sterile 15 mL tube and centrifuged at an RCF of 200×g for 5 min to pellet the cells. The supernatant was aspirated and the cell pellet was re-suspended in PBS by pipetting gently no more than 2-3 times.

hADSCs (female, 38Y and 44Y, PT5006, lot nos. 0000421627 and 0000692059, respectively; Lonza Walkersville, Inc., Walkersville, MD, USA) were used in this study; 38Y and 44Y indicate the donors’ age in years. The hADSCs were seeded in a 75 cm² flask with 15 mL of medium prepared from a medium kit (PT-4505 ADSC BulletKit™, Lonza Walkersville, Inc.) and maintained at 37 °C in a humidified 5% CO₂ atmosphere. The medium was changed every 3 or 4 days; the cells were passaged at approximately 90% confluency, and passages 3, 4, or 5 (4, 5, or 6 after cell preparation) were used for experiments. Cells were washed with phosphate-buffered saline without calcium and magnesium and trypsinized with trypsin/EDTA solution (CC-5012, Lonza Walkersville, Inc.) for 3-5 min at 37 °C. The trypsin reaction was stopped by addition of trypsin-neutralizing solution (CC-5002, Lonza Walkersville, Inc.), and hADSCs were subsequently detached by pipetting. The supernatant containing hADSCs was transferred to a 50 mL conical tube and centrifuged at 210 × g for 5 min at room temperature. The supernatant was aspirated, and hADSCs were resuspended in LR for use in storage experiments.