Cell Proliferation ELISA, BrdU (chemiluminescent) Protocol & Troubleshooting

Product No. 11669915001

Protocol

Calculation of a stimulation index

To calculate a stimulation index, first calculate the average of duplicates or triplicates, and subtract the background value (without cells) from each sample and control. Sample values (stimulated cells) are then divided by values obtained for the negative control (untreated samples). This results in unitless relative values which can be compared between different tests. The definition of a significant stimulation is determined empirically for each experimental setup, and strongly depends on cell type, type of stimulus, and other factors.

Troubleshooting

Low signal intensity for lymphocytes

To study the proliferation of lymphocytes, cells are stimulated, e.g., with growth factors, cytokines or mitogens. The increase in cell numbers can lead to cluster formation of the lymphocytes: Cells from the same progenitor can stick together and form aggregates in the culture plate. This effect can disturb antibody recognition of the ELISA system and result 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 uniform accessibility of each cell during antibody recognition of the BrdU-label.

This aggregation 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, carefully trypsinize the cells to generate single cells, centrifuge the microplate, and dry the cells as described in the package insert for suspension cells. Proceed using the standard procedure.

Materials

     
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