( ...)Quantitative Measurement of Cell Proliferation,,,Using the BrdU ELISA: A Comparison,,,Between Colorimetric and Chemiluminescent Detection

Quantitative determination of DNA synthesis in cell cultures is now a routine procedure in many laboratories. Protocols are available for various applications, especially in cell culture systems. The effects of growth factors, inhibition of cell division by exogenous factors, stimulation/inhibition by cytokines, development of serum-free media, influence of hormones and receptor activity on proliferation, and selection advantages for aneuploid cells in vitro are just a few examples where the determination of DNA synthesis provides important information.

In the past, radioactive tagging of newly synthesized DNA with 3H-labeled thymidine was the most frequently applied method, while today non-radioactive labeling with BrdU, a thymidine analog, is finding increasing use. Although the advantages of non-radioactive immune techniques using BrdU or anti-BrdU are not disputed, the scope and limitations of the available systems for detecting BrdU are not always clearly defined. These practical aspects have been examined in more detail in the experiments described below. Besides enabling microscopic detection at the cellular level, the ELISA technique represents a method for routine screening. Two different detection systems are available: a) the conventional colorimetric test and b) a chemiluminescence assay also based on the anti-BrdU technique. One of these test methods will be suitable for use depending on the problem and the available laboratory facilities.

We are particularly interested in the cultivation of human prostatic epithelial cells through several passages. For this purpose, a special culture medium containing exogenous growth factors and added hormones was developed [1]. In the course of this work we have examined the colorimetric and the chemiluminescence variants of the BrdU ELISA (Roche Molecular Biochemicals) in a series of parallel experiments.

Materials and Methods

Details of our special cell culture system and a short review on current measurement techniques for cell proliferation has been published [1-4]. We have investigated the proliferation of two distinct human prostatic carcinoma cell lines, P1/289 and P1/373 previously established in our laboratory.

In preliminary experiments, we had already defined the relevant cultivation and labeling intervals as well as the optimal cell concentrations for these cells. We have now investigated the influence of growth factors (epidermal growth factor, EGF) as well as the effects of insulin (Ins) and hydrocortisone (HC) on DNA synthesis in the cell lines P1/289 and P1/373. For this experiment, 104 cells were seeded in standard 96-well microtiter plates in a volume of 200 l medium/well and cultivated in complete medium (DMEM/F12, Life Technologies) containing 10 % fetal calf serum (FCS) supplemented with EGF (10 ng/ml), hydrocortisone (10 ng/ml), cholera toxin (CT, 10 ng/ml), and insulin (4 g/ml). To examine the effects of the additives (DMEM/F12+) on cell proliferation, the cell lines were also cultivated in parallel in their absence (DMEM/F12-).

A second application concerned another cell culture model: To prevent the outgrowth of fibroblasts when establishing cell cultures from heterogeneous tissue samples, cultivation of epithelial cells in the presence of cholera toxin is recommended. When added to normal cell culture medium, this toxin selectively suppresses the growth of fibroblasts. Accordingly, we cultured human fibroblasts obtained from explants (ZF 07) in microtiter plates. The rate of DNA synthesis in standard medium (DMEM) was determined and compared with that in a medium containing 10 ng/ml cholera toxin (Sigma) for inhibition of fibroblast growth (DMEM + CT).

In a third set of experiments, we examined the advantages of a serum-free, special medium (PromoCell) for the cultivation of human umbilical vein endothelial cell (HUVEC) in comparison with the standard medium (RPMI + 10 % FCS).

In each case, 24 hours after seeding of the cells, BrdU was added to a final concentration of 1 M. After incubation for further 24 hours, DNA synthesis was assayed with the Cell Proliferation ELISA, BrdU (Roche Molecular Biochemicals) using colorimetric or chemiluminescence detection, both according to the manufacturer's instructions. Newly synthesized BrdU-DNA was determined using an ELISA reader (BioRad, 450) or a luminometer (Orion, Berthold Detection Systems).

Results

The results clearly show that, depending on the labeling conditions or the proliferation behavior of the cells, widely differing results are obtained with the two detection systems, colorimetry and chemiluminescence (Figure 1).



In the cases of the prostatic carcinoma cells lines P1/289 and P1/373 treated with hormone, growth factor, and toxin, an increase in the DNA labeling index of more than 50 % is observed in the chemiluminescence test whereas, for the same cells, the colorimetric BrdU assay reveals an increase of only about 20 %. Growth inhibition by cholera toxin in fibroblast cultures results in a significant reduction of BrdU incorporation from 100 % in the control to values of 44 % and 35 % in the presence of 10 ng/ml cholera toxin, irrespective of the detection system used.

Comparable results for changes in proliferation behavior, expressed as the amount of newly synthesized DNA, were also obtained with the two detection systems in comparative studies on cultivated human umbilical vein endothelial cells. In this case, the serum-free medium from PromoCell was clearly advantageous for proliferation, showing an increase of almost 50 % in comparison with the RPMI standard medium, irrespective of whether the colorimetric assay (+50 %) or the chemiluminescence assay (+49 %) was used.

Discussion

The results show that conventional colorimetric tests exhibit a limited linear range. In general, this linearity is found in the absorption range between 0.5 and 1.5. Although most readers can also detect higher absorption values (sometimes up to 3.0), linear relationships between the measured absorption values and the actual incorporation of BrdU do not exist in these ranges. Accordingly, if one wants to document an increase in proliferation or inhibition of a cell line by exogenous growth factors, there are only two possibilities:

a) To perform the measurements in a region lying within the limits of the linearity range. This neccesitates appropriate preliminary trials.

b) To use a test exhibiting a linearity range extending over several orders of magnitude.

Overall, the use of chemiluminescence test systems is advantageous since the linearity region covers around 8-4 orders of magnitude and thus also provides the possibility of detecting and documenting extreme changes of the DNA synthesis index in one and the same experiment. The use of an extensively supplemented medium for the cultivation of human prostatic carcinoma cell lines may, at first, seem to be unnecessary on the basis of the BrdU colorimetric data as increases of merely 16 % and 20 % were recorded. However, the extent of the cell proliferation already leads to values in the non-linear region. Use of the chemiluminescence technique results in different sets of data. In this case, depending on the cell line, growth advantages of 55 % or 63 % are achieved by supplementation of the medium. The alternative, to choose the linear region of the colorimetric test for the experiments, is in general not very practicable since the problems investigated with these test systems usually involve examination of the effects of several parameters in a single experiment. The positive or negative changes in the proliferation index or incorporation of BrdU may vary from parameter to parameter. Thus, it is often necessary to repeat one or more of the experiments, in some circumstances with differing cell numbers or labeling times.

The colorimetric test can be employed without difficulty in cell systems and experiments in which short labeling times are chosen or in which changes in proliferation are measured at the end of a growth curve, near confluence. This is shown by the results with comparatively slowly growing fibroblasts (ZF 07) and the endothelial cells (HUVEC). In these cases, the two BrdU systems provide almost the same information, namely inhibition of cell division of fibroblasts upon use of cholera toxin or the advantage of serum-free endothelial medium in comparison with a standard cell culture medium. The absorption values of the colorimetric BrdU test are in the linear region; thus the differences in BrdU incorporation can be reliably recorded by instruments and are comparable with those from the chemiluminescence technique.

When measurements have to be made on several differently proliferating cells under different conditions in one experiment, the chemiluminescence test is the method of choice because it enables linear results to be obtained over a wide interval. When, on the other hand, proliferation measurements are made on only a few or even just one cell line over a limited period of time, the more easyto- use colorimetric test can be employed. Using a colorimetric assay one must always consider that this test has only a limited linear range. In contrast to numerous other ELISA kits, however, when the linear range of the BrdU ELISA is exceeded, it is not possible to just dilute the sample because the latter is a cellular ELISA in which the labeled, cultivated cells produce the signals. In such cases, the only alternative is to perform a completely new experiment with a shorter labeling time. Reducing the cell number in order to bring the signal to the linear range is not feasible because this would change the overall proliferation behavior of the cell population in vitro.

These observations are of practical relevance. Not all laboratories performing routine DNA synthesis determinations possess the apparatus required for luminescence measurements because it is considerably more expensive than systems for measuring absorption. The experiments described here may help in this decision-making process.




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