Name: measuring proliferation of vascular smooth muscle cells using click chemistry
Uploaded: 2019-10-30T13:00:00
Description: Watch this Scientific Journal Video about Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry at JoVE.com

We thank Katie Carroll for technical assistance. We also thank Dr. David Cool and the Proteomics Analysis Laboratory for instruction on and provision of the Cytation imaging plate reader. This work was supported by a Wright State Foundation grant (to L.E.W.).

1. Detection of EdU using fluorescent label
<ol>
	<li>Stock solutions
	<ol>
		<li>Prepare a 5 mM EdU stock solution by adding 12.5 mg of EdU to 10 mL of double distilled H2O.</li>
		<li>Prepare the labeling solution components: 200 mM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) (100 mg in 1.15 mL H2O), 100 mM CuSO4 (15.95 mg in 1 mL H2O; make fresh), 10 mM Cy3 picolyl-azide (1 mg in 95.3 mL H2O, stored in 5 &#181;L aliquots at -20 &#176;C), and 1 M sodium asc...

<ol>
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Incorporation of EdU is a simple, straightforward way to measure cell proliferation; it is particularly useful for adherent cells13. Our protocol uses smooth muscle cells, but it is applicable to any adherent cell (epithelial, endothelial, etc.). Although the protocol is not complicated, the one critical step is making the labeling solution &#8212; the ingredients must be added in the order listed. In addition, like chemiluminescent Western blots, if using the luminescence readout the plate must b...

Proliferation is a critical part of cellular function1,2. Control of proliferation influences normal processes such as development, and pathologic processes such as cancer and cardiovascular disease. Hyperplastic growth of vascular smooth muscle cells, for example, is thought to be a precursor to atherosclerosis3. Changes in cell proliferation are also used to assess potential toxicity of new drugs. Given its widespread impact, measuring proliferation is a mainstay of many cell biology-based laboratories.
Cell proliferation can be measured by simply counting cells if the cell population of interest divides fairly rapidly. For slower growing cells, cell counts may be less sensitive. Proliferation is often measured by incorporation of labeled nucleoside analogs. Although the gold standard is 3H-thymidine incorporation, many laboratories are getting away from this method given the availability of newer, non-radioactive alternatives. These include cytoplasmic fluorescent dyes, detection of cell cycle associated proteins, and incorporation of non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU)4.
Cytoplasmic dyes, such as carboxyfluorescein diacetate succinimidyl ester (CFSE), detect proliferation because the intensity of the dye halves each time the cell divides5. This technique is commonly used in flow cytometry for non-adherent cells. It has not been used much for adherent cells, but with the new generation of imaging plate readers this may change. Detection of cell cycle associated proteins through antibody-based techniques (flow cytometry, immunohistochemistry, etc.) is often used for tissues or non-adherent cells. These cells/tissues must be fixed and permeabilized prior to staining. Nucleoside analogs BrdU and EdU are similar in approach to 3H-thymidine, but without the inconvenience of radioactivity. Incorporation of BrdU is detected by anti-BrdU antibodies, and therefore cells must be fixed and permeabilized prior to staining. In addition, cells must be treated with DNase to expose the BrdU epitope.
Incorporation of EdU is detected by click chemistry, in which alkyne and azide groups &#8220;click&#8221; together in the presence of catalytic copper. Either group can serve as the biosynthetically incorporated molecule or detection molecule4. Commercially available nucleoside analogs have the alkyne group attached. For proliferation assays, therefore, the alkyne functionalized nucleoside analog is detected by an azide conjugated to a fluorescent dye or other marker. Incorporation of EdU has several advantages over BrdU. First, because alkyne and azide groups are not found in mammalian cells, this interaction is highly specific with a low background6. Because both groups are small, cells do not need to undergo DNA denaturation to expose the nucleoside analog as required for BrdU7. Finally, click chemistry is highly versatile, and can be used for the metabolic labeling of DNA, RNA, protein, fatty acids, and carbohydrates8,9,10,11. If the metabolically labeled target of interest is on the cell surface, live cells can be labeled12. In addition, cells can be further processed for immunofluorescent staining with antibodies.
The following protocol describes the use of EdU incorporation and click chemistry to measure proliferation in human vascular smooth muscle cells (Figure 1). We show three different ways incorporation of EdU can be measured, representative results from each, and their advantages and disadvantages. Measuring proliferation via click chemistry is particularly useful for adherent, slower growing cells. In addition, the morphology of the cell is well maintained and antibody-based staining can also be performed.

<table>
	<tbody>
		<tr>
			<td>5-Ethynyl-2&#39;-deoxyuridine</td>
			<td>Carbosynth</td>
			<td>NE08701</td>
			<td></td>
		</tr>
		<tr>
			<td>biotin picolyl azide</td>
			<td>Click Chemistry Tools</td>
			<td>1167-5</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>Fisher Scientific</td>
			<td>C1297-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 picolyl azide</td>
			<td>Click Chemistry Tools</td>
			<td>1178-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation Plate Reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS Microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>Na ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>11140-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue Fixed Cell Stain Ready Probes</td>
			<td>Invitrogen</td>
			<td>R37606</td>
			<td>DAPI nuclear stain</td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer</td>
			<td>Li-Cor</td>
			<td>927-40000</td>
			<td>blocking buffer</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Services</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>SH3002802</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Life Science</td>
			<td>S3014-5kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Horseradish Peroxidase Conjugate</td>
			<td>Life Technologies</td>
			<td>S911</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Signal ELISA Femto</td>
			<td>Thermo Scientific</td>
			<td>PI137075</td>
			<td>ELISA substrate</td>
		</tr>
		<tr>
			<td>Synergy Plate Reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>THPTA</td>
			<td>Click Chemistry Tools</td>
			<td>1010-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Hydroxymethyl Aminomethane Hydrochloride (Tris-HCl)</td>
			<td>Fisher Scientific</td>
			<td>BP153-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X 100</td>
			<td>Bio-Rad</td>
			<td>1610407</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring proliferation of vascular smooth muscle cells using click chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

In Figure 2, we demonstrate the outcomes of three different experiments measuring proliferation of VSMC in response to PDGF. After growing the cells in media formulated for SMCs, we carried out the experiment in serum free DMEM, to eliminate any potential effects of serum or growth factors on proliferation. In Figure 2A we compare results, from the same experiment, using a fluorescent vs luminescent readout. To read these plates, we used a multimode microplate r...

Watch this Scientific Journal Video about Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry at JoVE.com

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...

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.

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