Figure 1. Measurements of extracellular ATP after nerve cord injury. (A) An ATP luminescence assay (luciferin/luciferase) was used to measure the increase in extracellular ATP after nerve cord crush. Connectives were dissected and kept overnight in supplemented L-15. No (uncrushed control), one, two, or three crushes were made to the connectives that were then incubated at 18°C for 30 min in 150 µl of leech Ringer’s solution. 100 µl of the supernatant was analyzed for its ATP content. Connectives with two and three crushes showed statistically significant increases in extracellular ATP compared with the uncrushed control (*, P < 0.05; n = 3). (B) Extracellular ATP was elevated for at least 2 h after injury. Samples (100 µl) of supernatant of two connectives each crushed twice (total volume 150 µl) were collected at 15, 30, and 120 min after making the crushes, and samples were analyzed using a luciferin/luciferase luminescence ATP assay. Significant elevation of extracellular ATP remained for at least 2 h after injury (*, P < 0.05; n = 3). (C and D) Release of ATP >15 min after injury and the effect of 10 µM CBX. Connectives were crushed twice, incubated for 15 min at room temperature, washed three times with Ringer’s solution, resubmerged in 150 µl of fresh Ringer’s solution, and incubated at room temperature for 30 min. 100-µl aliquots of supernatant were analyzed with luciferin/luciferase luminescence assay to measure levels of extracellular ATP. In C, ATP was elevated 15 min after the injury (P < 0.05; n = 3). In D, in another experiment, the continued release of ATP was attenuated by treatment with 10 µM CBX. Control and CBX conditions are displayed as percentages of values for uncrushed tissues to control for the direct effect of CBX on the luciferase assay, for CBX may artificially increase the absolute luminescence values slightly (P < 0.05; n = 3).
Figure 1.

Measurements of extracellular ATP after nerve cord injury. (A) An ATP luminescence assay (luciferin/luciferase) was used to measure the increase in extracellular ATP after nerve cord crush. Connectives were dissected and kept overnight in supplemented L-15. No (uncrushed control), one, two, or three crushes were made to the connectives that were then incubated at 18°C for 30 min in 150 µl of leech Ringer’s solution. 100 µl of the supernatant was analyzed for its ATP content. Connectives with two and three crushes showed statistically significant increases in extracellular ATP compared with the uncrushed control (*, P < 0.05; n = 3). (B) Extracellular ATP was elevated for at least 2 h after injury. Samples (100 µl) of supernatant of two connectives each crushed twice (total volume 150 µl) were collected at 15, 30, and 120 min after making the crushes, and samples were analyzed using a luciferin/luciferase luminescence ATP assay. Significant elevation of extracellular ATP remained for at least 2 h after injury (*, P < 0.05; n = 3). (C and D) Release of ATP >15 min after injury and the effect of 10 µM CBX. Connectives were crushed twice, incubated for 15 min at room temperature, washed three times with Ringer’s solution, resubmerged in 150 µl of fresh Ringer’s solution, and incubated at room temperature for 30 min. 100-µl aliquots of supernatant were analyzed with luciferin/luciferase luminescence assay to measure levels of extracellular ATP. In C, ATP was elevated 15 min after the injury (P < 0.05; n = 3). In D, in another experiment, the continued release of ATP was attenuated by treatment with 10 µM CBX. Control and CBX conditions are displayed as percentages of values for uncrushed tissues to control for the direct effect of CBX on the luciferase assay, for CBX may artificially increase the absolute luminescence values slightly (P < 0.05; n = 3).

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