Cell Proliferation ELISA, BrdU (colorimetric) Protocol & Troubleshooting

Product No. 11647229001

Protocol

Measurement at different wavelengths

It is possible to measure the color development over a period of time without adding stop solution and measuring at 370/492 nM, to easily identify the optimal experimental conditions.

With addition of stop solution and measurement at 450/690 nM you can more easily measure the exact value after a given time point in the experiment.

Simultaneous use of Cell Proliferation Reagent WST-1 and Cell Proliferation ELISA BrdU, colorimetric

Figure 52a of the Roche ′Apoptosis, Cytotoxicity and Cell Proliferation Manual′, 4^th edition shows the combined use of the Cell Proliferation Reagent WST-1 and the Cell Proliferation ELISA BrdU, colorimetric for simultaneous measurement of cell viability and cell proliferation. A protocol is briefly described in the figure legend. To perform assays simultaneously, order both kits; additional information is provided in the corresponding package inserts.

Calculation of a stimulation index

To calculate a stimulation index, first calculate the average of duplicates/triplicates, and then substract the background value (without cells) of each sample and control. The values of samples (stimulated cells) are then divided by the negative control (untreated samples). This results in relative values (without any units), which can be compared between different tests. Concerning definition of a significant stimulation, this has to be determined according to the researchers experience for each experimental setup, since it is strongly depending on cell types, stimulus and other factors of influence.

Troubleshooting

Low signal intensity for lymphocytes

To study the proliferation of lymphocytes, the cells are stimulated, e.g., with growth factors, cytokines or mitogens. The increase in cell numbers can lead to cell cluster formation of the lymphocytes: Cells from the same progenitor stick together and form aggregates in the culture plate. This effect may disturb the antibody recognition of the ELISA system, resulting in an underestimation of the response. To avoid signal variation, carefully resuspend the cells by pipetting after the BrdU-labeling period and prior to removing the culture medium. This will enable equal accessibility of each cell for antibody recognition of the BrdU-label.

This problem mostly affects suspension cells, such as lymphocytes. Should you experience this problem with adherent cells, remove the labeling medium after the labeling period is completed, and carefully trypsinize the cells to produce single cells; centrifuge the microplate, and dry the cells, as described in the package insert for suspension cells; then perform the standard procedure.

Cell culture systems can differ greatly in cell number, proliferating activity and incubation periods, producing absorbance values that are either too high or too low.