Astressin 2b ast2b

Pre-impact treated explants were incubated at 37 °C and 5% CO₂ in media containing the relevant recombinant peptides or modifying agents in 1% FCS media for 30 min prior to impact. Post-impact treated explants were placed into fresh media with the relevant recombinant peptides or modifying agents added 30 min following impact. All explants were incubated for a further 72 h with no further media changes prior to assay.
In order to test the role of Ucn in the prevention of chondrocyte cell death, explants were incubated with 0.1 µM human Ucn (Ucn1) (Tocris, Bristol, UK) or Ucn2 (GenScript, Piscataway, NJ, USA). To investigate the role of Ucn receptors, CRF (Tocris, Bristol, UK), CP-154526 (CP) (Tocris, Bristol, UK) and Astressin 2B (Ast2B) (Tocris, Bristol, UK) were used at 0.1 µM. For ion channel identification, Yoda1 (Tocris, Bristol, UK) and GsMTx4 (Alomone Labs, Jerusalem, Israel) were also used at 0.1 μM concentration. HC-067047 (Tocris, Bristol, UK) was used either at 50 µM concentration, or in a concentration from 12.5 µM to 75 µM. Gadolinium (Gd³⁺) (Tocris, Bristol, UK) was used for calcium experiments at 500 µM concentration.