Methods Materials Unless otherwise specified, all reagents were from Sigma Aldrich Chemical Co, 10panx1 and its scramble control, the P2X7 inhibitors A438079 and AZ10606120, and GSK1016790A, were obtained from Tocris. Chondrocyte cultures Primary hyaline articular chondrocytes were isolated from knee joints of 3 to 5 year old pigs as previously described. Afatinib mw Knee cartilage was obtained from pigs slaughtered at a local abattoir and was used in accord ance with guidelines from the Subcommittee on Animal Use of the Research and Development Committee of the Zablocki VA Medical Center. Chondrocytes were plated in high density short term monolayer cultures and used within 3 days of plating. DMEM was used for all experiments. Initial experi ments were repeated with chondrocytes embedded in 2% agarose constructs.
To embed chondrocytes, freshly digested cells were mixed 1,1 with 4% agarose in Hanks Balanced Salt Solution. One hundred ul of warm agarose containing cells were added to each well of a 96 well plate and allowed to solidify. After solidification, 150 ul of DMEM were added to each well. eATP measurements Media were removed from Inhibitors,Modulators,Libraries chondrocytes plated in 96 well clear bottom black plates, and replaced with fresh serum free DMEM with or without ATP modulators or other additives. After 30 minutes aliquots of media were removed and replaced with an equal volume of sterile water to expose cells to hypotonic media or fresh DMEM as a control. After 10 minutes eATP levels were measured in the media using the Sigma ATP Assay Mix and read in Inhibitors,Modulators,Libraries a BioTek Synergy HT plate reader.
The osmolarities of all media prepa rations including those with Inhibitors,Modulators,Libraries and without inhibitors and other additives were measured with an Osmette osmom eter. Media Inhibitors,Modulators,Libraries osmo larities were as follows, undiluted media 362 Inhibitors,Modulators,Libraries to 302 mOsm L, 15% H2O 282 to 249 mOsm L, 35% H2O 216 to 192 mOsm L, and 50% H2O 166 to 143 mOsm L. No media additives, except for water, altered media osmolarity more than 10%. We chose to use 10 to 50% water as an osmotic challenge, as this level of osmotic stress typically induces eATP release in other cell types. Each culture additive and osmotic condition was tested for effects on the ATP standard curve. If effects were noted, as they were in the case of sodium pyrophos phate and Brilliant Blue G, calculated ATP levels were adjusted accordingly.
ATP metabolizing ecto enzyme activities Specific activities of the ecto enzymes that metabolize ATP were measured, as changes in these enzyme activities could affect eATP levels without altering transport. NTPPPH activity was measured using 2 mM p nitrophenol thymidine exactly monophosphate as a substrate. Briefly, the media were removed and replaced with PNPMP in HBSS. The cells were incubated for 2 h at 37 C and the reaction was stopped with the addition of 0. 1 N NaOH. The absorbance was measured at 410 nm using a Biotek plate reader.