By Grozier W.J., Federighi H.
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Extra resources for On the Measurement of Critical Thermal Increment for Biological Processes
1998) Bioluminescence detection of ATP release mechanisms in epithelia. Am J Physiol 275, C1391–C1406. 15. , and McElroy, W. D. (1978) Purification and properties of firefly luciferase. In Methods Enzymol 57, 3–15. Chapter 4 High-Throughput Quantitative Bioluminescence Imaging for Assessing Tumor Burden Angelina Contero, Edmond Richer, Ana Gondim, and Ralph P. Mason Abstract Bioluminescence imaging (BLI) has emerged during the past 5 years as the preeminent method for rapid, cheap, facile screening of tumor growth and spread in mice.
3. 4. At the end of each assay, an ATP–luminescence relationship (calibration curve) is generated to calculate ATP concentrations. 3. Measurement of Cell Surface ATP Concentrations Using Soluble Luciferase 1. Wash the surface of cultures with PBS,3 Â . 2. 5 cm cultures. Bilaterally for polarized cultures – 1 cc and 25–500 ml to luminal and serosal side, respectively, when 12-mm Transwell is used). Equilibrate the cultures in an incubator (37°C and 5% CO2) for 1 h. 3. 2. 4. Notes 1. Firefly luciferase catalyzes the following reaction: D-luciferin þ ATP þ luciferase (L) !
Incubate for 1 h on ice. 4. Wash 3 Â with PBS. 5. 5 mg/mL purified SPA-luc (see Note 4) for 1 h at 4°C in the dark. SPA-luc will bind to the Fc domain of the antibody attached to the cells, as indicated in Step 3. 6. Wash carefully 3 Â with PBS. , HBSS/HEPES). Keep cultures in the dark at RT for 30 min to equilibrate the extracellular ATP concentrations. 2. Measurement of Cell Surface ATP Concentrations Using SPA-luc 1. Place a SPA-luc-bound cell culture in the Turner TD-20/20, add soluble luciferin (150 mM final, to the apical solution for polarized cultures) and close the lid.