The overall goal of this procedure is to use autonomously by luminescent human cells for continuous and real-time cytotoxicity monitoring. This is accomplished by first obtaining a sufficient number of actively growing auto bioluminescent cells. In the second step, equal numbers of cells are seeded into individual wells of a multi well plate.
Next, the cells are treated with a compound of interest over a range of concentrations. In the final step, the bioluminescent signal is measured using a compatible instrument. Ultimately, the bioluminescent output dynamics can be used to demonstrate the effect of the test compound on the metabolic activity of the cells.
The main advantage of this technique over existing methods like those using luciferase isolated from insects or submarine species, is that no sample destruction or substrate addition is required to produce a detectable signal. Demonstrating the procedure will be James Webb, an undergraduate student from a laboratory After growing the bioluminescent cells of interest to 80%confluence in the appropriate culture medium, wash the cell culture in PBS with gentle swirling next trypsin eyes, the cells at 37 degrees Celsius until the cells have detached from the flask. Then collect the resuspended cells in PBS, transfer them into a clean centrifuge tube and determine the number of cells after counting centrifuge the cell suspension for five minutes at 300 Gs at room temperature and resus.
Suspend the pellet in fresh prewarm medium, then seed equal numbers of cells into individual wells of an opaque multi-well plate plate, an equal volume of medium alone in one set of triplicate wells as a negative control. Then leaving three wells of cells untreated as controls at the experimental compound of interest directly to the cells at the appropriate experimental concentrations in triplicate. Immediately following the experimental treatment, place the plate in the imaging chamber of the appropriate instrument for image acquisition and bioluminescence measurement.
Then set the appropriate integration time for each reading and measure the bioluminescence every 15 minutes over a 24 hour period. Finally, use the appropriate compatible software to identify a region of interest in each well. To quantify the bioluminescent intensity for each experimental sample, display the light output in photons per second to illustrate the total flux or in photons per second per square centimeter per Ceridian, to demonstrate the average radiance of each sample.
In this study, the dynamics of auto bioluminescent HEC 2 9 3 cells were monitored continuously over a 24 hour period in response to antibiotic exposure. The toxic effects of this antibiotic, which is a member of the Blio Mycin family, known to kill living cells by binding two and cleaving DNA, were demonstrated via a decrease in bioluminescent production compared to untreated cells, which can be directly visualized through pseudocolor images. As shown here, the bioluminescent nature of HEC 2 9 3 cells permits the repeated parallel screening of samples under varying treatment conditions, allowing the comparison of different experimental treatment concentrations at any given time point for an easy dose response analysis.
For example, in this graph, the bioluminescence produced by the cells after 15, 18, or 20 hours of treatment was plotted against the concentrations of the experimental compound added illustrating that increasing the antibiotic treatment results in the reduction of bioluminescent production by the cells in a dose dependent manner. The technique presented in this video, it's suited for tracking acute toxic effects, but it can be adapted to assess slow acting or long-term toxicity by repeatedly imaging the cells at increased time intervals while maintaining the culture under standard incubation conditions between readings.
