Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

Proliferation is an important measure of cell function. This protocol allows investigators to develop a sensitive proliferation assay in their labs without the high cost of commercially available kits. The main advantage of this technique is that it avoids harsh treatment of cells and/or the use of radioactivity.

Demonstrating this technique will be Vicky Wong, the manager of my laboratory. To assay proliferation, remove vascular smooth muscle cells from a flask with smooth muscle cell media in 2%fetal bovine serum and plate at 20, 000 cells per milliliter in DMEM in a 96-well plate. Grow the cells at 37 degrees Celsius overnight.

Add 30 nanograms per milliliter platelet derived growth factor to three to six wells as a positive control to stimulate proliferation. Then add PBS to the same number of wells as a negative control. Incubate for 72 hours.

Dilute previously prepared five millimolar EdU stock to one millimolar and add two microliters to each well for the last 24 hours of the 72 hours culture period. Keep one set of replicates without EdU to determine background fluorescence and luminescence. Twenty-four hours later, fix the cells by removing media, then adding 150 microliters per well of 4%paraformaldehyde and incubate 10 minutes at room temperature.

Remove the paraformaldehyde and add 150 microliters of 1%TX-100 in water per well and incubate at room temperature for 30 minutes. Wash three times with PBS to remove the TX-100. To detect incorporated EdU using fluorescence, make 10 milliliters of labeling solution per 96-well plate just prior to use by adding reagents from stock solutions in the right order:20 microliters THPTA, 20 microliters copper sulfate, five microliters fluorescein picolyl-azide, and 100 microliters sodium ascorbate into PBS for a total volume of 10 milliliters.

The most critical step is to make the labeling solution just prior to use. Remove PBS from wells, add 150 microliters of labeling solution to each well and incubate 30 minutes at 37 degrees Celsius. To detect all nuclei, add DAPI to each well and image on a fluorescent microscope or imaging plate reader.

To avoid manual counting and when an imaging plate reader is not available, this protocol can be modified to a luminescence readout using HRP azide and an ELISA substrate. To detect EdU using luminscence, first prepare tris-buffered saline with 0.1%polysorbate 20. Then add 150 microliters of blocking buffer to each well and incubate for 90 minutes at room temperature.

After removing the blocking buffer, wash the cells three times with 200 microliters of PBS. To detect incorporated EdU, first prepare 10 milliliters of labeling solution per 96-well plate by adding reagents from stock solutions in the right order. First, 20 microliters THPTA, then 20 microliters copper sulfate, five microliters biotin picolyl-azide, 100 microliters sodium ascorbate into PBS for a total volume of 10 milliliters.

Wash wells five times three minutes with 200 microliters of TBST with shaking. Quench endogenous peroxidases with 150 microliters of 0.3%hydrogen peroxide for 20 minutes at room temperature. After removing hydrogen peroxide, wash five times three minutes with 200 microliters of TBST with shaking.

Add 50 microliters of diluted streptavidin-horseradish peroxidase in TBST to each well and incubate at room temperature for one hour. Wash five times three minutes with 200 microliters of TBST with shaking. Add 50 microliters of chemiluminescent ELISA substrate to each well and read immediately in a plate reader capable of detecting luminescence.

Although fluorescent nuclei can be detected by a standard fluorescence plate reader, using the luminescence protocol, the fluorescent scan is converted to a homogenous liquid readout detected with streptavidin-horseradish peroxidase and a standard ELISA substrate. The advantage of this method compared to a fluorescent readout is a more homogenous readout and the plate can be read in seconds as seen here when proliferation of VSMC in response to PDGF was measured. The disadvantage is that the background is higher than with fluorescence, thus negative controls tend to be higher.

In an attempt to decrease variability, results were normalized to initial cell counts. However, in a separate set of experiments, it was shown that this method is not sensitive enough to detect small differences in cell numbers. The most efficient and accurate way to count EdU positive and total cells is by using an imaging plate reader.

If this type of plate reader is not available, manual counting will also yield accurate results. When comparing these two methods, the EdU positive manual count was identical to the automated count as were the total unstimulated counts. The advantage of these two methods is visibility of the cells to be counted, improved accuracy, and that nonspecific staining of debris can be seen and avoided.

The main disadvantage with the manual counting method is time. Remember to make the labeling solution fresh just prior to use and add the ingredients in the order provided in the written protocol. If intensity of staining is dim, try adjusting the chelator to copper ratio.

Remember that paraformaldehyde is toxic. It should be used in a fume hood while wearing gloves. The procedure itself is not difficult but may need to be optimized by adjusting chelator to copper ratios in the labeling solution.

In addition, incubation times and length of EdU treatment can be adjusted depending on the cell type and the specific question being asked. This technique is compatible with cell surface and intracellular staining so that the characteristics of dividing and non-dividing cells can be determined. Although commonly used to measure proliferation, click chemistry can also be used for the metabolic labeling of RNA, proteins, fatty acids and carbohydrates.

If the metabolic target of interest is on the cell surface, live cells can be labeled.