For all ex vivo experimental procedures, murine femora were submerged in iso-osmotic (300mOsm) wash medium (Dulbecco’s Modified Eagle Medium (DMEM)-High Glucose) (Invitrogen; Grand Island, NY, USA). During isolation, in vitro chondrocytes were maintained in iso-osmotic (380mOsm, to account for the loss of proteoglycan molecules [20 (link)–22 ]) feed medium (DMEM/F-12) (Invitrogen) containing 10% Fetal Bovine Serum and 2% Pen/Strep. During dye incubation and all confocal microscope imaging, in vitro chondrocytes were submerged in wash medium augmented with 1% Kanamycin, 0.5% Fungizone and 0.1% Gentamycin. All media was adjusted to pH 7.4.
During confocal imaging, chondrocytes were presented with a hypo-osmotic (−50 or −100mOsm) challenge, hyper-osmotic (+50 or +100mOsm) challenge, or 10nM GSK1016790A (GSK101, Sigma, Oakville, ON) a potent activator of TRPV4 [23 (link)]. Appropriate control experiments, iso-osmotic (final osmolarity 300mOsm ex vivo or 380mOsm in vitro) and GSK vehicular control (Dimethyl sulfoxide, Fisher Scientific, Ottawa, ON) 1:106)), were also conducted. Medium osmolarity was adjusted using distilled water/sucrose, and confirmed using a freezing point osmometer (Advanced Instruments; Norwood, MA, USA).
During confocal imaging, chondrocytes were presented with a hypo-osmotic (−50 or −100mOsm) challenge, hyper-osmotic (+50 or +100mOsm) challenge, or 10nM GSK1016790A (GSK101, Sigma, Oakville, ON) a potent activator of TRPV4 [23 (link)]. Appropriate control experiments, iso-osmotic (final osmolarity 300mOsm ex vivo or 380mOsm in vitro) and GSK vehicular control (Dimethyl sulfoxide, Fisher Scientific, Ottawa, ON) 1:106)), were also conducted. Medium osmolarity was adjusted using distilled water/sucrose, and confirmed using a freezing point osmometer (Advanced Instruments; Norwood, MA, USA).