07 Lider Disk
Dibe Naro
Cartilage explants were incubated with a fixed amount of 125I-TNFα and graded amounts of unlabeled TNFα. A. Equilibrium uptake ratio in bovine cartilage disks of 125I-TNFα (49.2 pM = 838 pg/ml), incubated in DMEM with 1% ITS, and 0.1% BSA at 37C for 48 hr in the absence (closed circle, viable cells) or presence (open square, non-viable cells) of 0.01% NaN3 and protease inhibitors. Mean SEM (n = 6 disks per condition, harvested from 2 joints). B. Equilibrium uptake ratio in adult human knee cartilage disks of 125I-TNFα (112 pM = 1910 pg/ml), incubated in 1PBS with 0.1% BSA, 0.01% NaN3 and protease inhibitors at 37C for 48 hr. Mean SEM (n = 8 disks for each condition, harvested from the distal femur of a 44 year old, male grade 0-1 joint).
Untreated 125I-TNFα (23 pM = 390 pg/ml) was incubated with or without untreated unlabeled TNFα (20 nM = 340 ng/ml). Cross-linked 125I-TNFα (81 pM = 1.38 ng/ml) was incubated with graded amounts of cross-linked unlabeled TNFα. The uptake ratio of cross-linked 125I-TNFα was not affected by varying the concentration of cross-linked unlabeled TNFα (1-way ANOVA, p = 0.23). Cartilage disks were incubated in 1PBS with 0.1% BSA, protease inhibitors, and 0.01% NaN3 for 24 hr at 37C. Mean SEM (n = 6 disks for each condition, harvested from 2 joints).
Bovine calf cartilage disks were either untreated (Control) or treated with 0.1 mg/ml trypsin for 48 hr at 37C (Trypsin) prior to incubation with 125I-TNFα (23 pM = 390 pg/ml) in the absence or presence of unlabeled TNFα (20 nM = 340 ng/ml) for 48 hr in 1PBS with 0.1% BSA, protease inhibitors, and 0.01% NaN3 at 37C. The measured uptake ratio was only affected by addition of unlabeled TNFα and not by trypsin (2-way ANOVA, p < 0.0001 for the effect of unlabeled TNFα; p = 0.164 for the effect of trypsin). Mean SEM (n = 5 disks for each condition, harvested from 1 joint).
08ab062aa8