Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

Summary

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.

Abstract

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’ deoxyuridine (BrdU) and 5-ethynyl-2′-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.

Introduction

Proliferation is a critical part of cellular function^1,2. 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’ deoxyuridine (BrdU) and 5-ethynyl-2′-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 “click” 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 carbohydrates^8,9,10,11. 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.

Protocol

1. Detection of EdU using fluorescent label Stock solutions Prepare a 5 mM EdU stock solution by adding 12.5 mg of EdU to 10 mL of double distilled H2O. 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 µL aliquots at -20 °C), and 1 M sodium asc…

Representative Results

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…

Discussion

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 — the ingredients must be added in the order listed. In addition, like chemiluminescent Western blots, if using the luminescence readout the plate must b…

Materials

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