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4. Prepare XTT labeling mix according to instructions and add 50 l to each well. Incubate at 37C for 1, 2, and 3 hr.
5. After 1 hr of incubation, place the plate into the Benchmark Plus.
6. To determine the optimum wavelength for the assay, run a spectral scan. In Microplate Manager PC software, select File>New Endpoint Protocol>Advanced Options>Scan Well. Scan three wells with different cell densities a cross the wavelength range 340800 nm with a step size of 5 nm. Next, construct a graph to show the peak wavelength around 475 nm (the re commended wavelength for reading the assay) for all samples scanned.
7. After the spectral scan, open the Microplate Manager software application. Under the File menu, select New Endpoint Protocol. Next, select the 96-well format and edit the template, selecting the wells used. Specify a single-wavelength reading at the peak wavelength determined in step 6.
8. Assay the plate using the Benchmark Plus by clicking the Run button. Repeat for the 2 and 3 hr incubation times.
9. After reading the samples, export or copy and paste the data points into Microsoft Excel software and plot them.
Results
Figure 1 shows the results obtained by scanning sample wells with different
cell densities (step 6 of Procedure). The scan resulted in an absorbance
curve that peaked at about 475 nm, the wavelength specified for sample
reading in the XTT kit instructions.
The assay was performed at 475 nm. Figure 2 shows a proportional increase in the OD value at 475 nm with increasing cell number.
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