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

1. Stock solutions
   1. Prepare a 5 mM EdU stock solution by adding 12.5 mg of EdU to 10 mL of double distilled H₂O.
   2. 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 ascorbate (200 mg in 1 mL H₂O; make fresh).
   3. Prepare resazurin stock solution at a concentration of 0.15 mg/mL. Filter sterilize and store 1 mL aliquots at 4 °C.
      NOTE: Picolyl azide is available conjugated to several different fluors, biotin, and horseradish peroxidase (HRP).
2. Label vascular smooth muscle cells (VSMC) with EdU.
   NOTE: Human VSMCs were isolated from iliac arteries via outgrowth from explanted pieces of tissue. When grown in serum free media with insulin, transferrin, selenium these cells express VSMC markers smoothelin, smooth muscle myosin heavy chain (SM-MHC) and SMC αactin¹³.
   1. Plate vascular smooth muscle cells at 2 x 10⁴/mL in Dulbecco’s modified Eagle’s medium (DMEM) in a 96 well plate.
      NOTE: Cells are grown at 37 °C in smooth muscle cell media with 2% fetal bovine serum.
   2. (Optional) Allow several hours to overnight for adherence. Add 20 µL resazurin stock to each well. Incubate at 37 °C for 3 h. Read the fluorescence signal in a plate reader (560_ex, 594_em). Replace media with fresh DMEM.
      NOTE: This step is optional. Resazurin is used to normalize cell numbers and account for well-to-well variability in plating¹⁴.
   3. Add phosphate buffered saline (PBS) or platelet derived growth factor (PDGF) 30 ng/mL to 3−6 wells per treatment at the beginning of the culture period and incubate for 72 h.
   4. Dilute 5 mM EdU stock to 1 mM. Add 2 µL to each well (final concentration 20 µM) for the last 24 h of the 72 h culture period. Do not add EdU to one set of replicates. These wells will be used to determine background fluorescence/luminescence.
      NOTE: The duration of culture and labeling with EdU is optimized for smooth muscle cells but may need to be modified per cell type.
3. Fix cells in paraformaldehyde (PFA).
   1. Remove media and add 150 µL of 4% PFA (in PBS) per well for 10 min at room temperature.
   2. Remove PFA and add 150 µL of 1% polyethylene glycol tert-octylphenyl ether (TX-100) (in water) per well for 30 min at room temperature.
   3. Remove TX-100 by washing three times with PBS.
      CAUTION: PFA is toxic, handle with care.
      NOTE: The protocol can be paused here. Fixed cells can be stored in PBS at 4 °C.
4. Detect incorporated EdU.
   1. Make labeling solution (10 mL per 96 well plate, using inner 60 wells) just prior to use by adding reagents from stock solutions in the following order: THPTA 20 µL, CuSO₄ 20 µL, Cy3 picolyl azide 5 µL, Na ascorbate 100 µL to PBS for a total volume of 10 mL.
   2. Remove PBS from wells and add 150 µL of labeling solution to each well. Incubate 30 min at 37 °C. To detect all nuclei, add 4′,6-diamidino-2-phenylindole (DAPI) to each well. Read on a fluorescence plate reader (Cy3 550_ex, 570_em; DAPI 350_ex, 470_em) or image on a microscope.
      NOTE: The protocol can be paused here. Fixed and stained cells can be stored in PBS at 4 °C and imaged at a later time.

2. Detection of EdU using luminescence

1. Complete sections 1.1–1.3.
2. Prepare Tris-buffered saline with 0.1% polysorbate 20 (TBST).
3. Block wells.
   1. Add 150 µL of blocking buffer (Table of Materials) to each well and incubate for 90 min at room temperature.
   2. Remove blocking buffer and wash three times with 200 µL of PBS.
4. Detect incorporated EdU.
   1. Make labeling solution as directed in step 1.4.1, substituting biotin picolyl azide for Cy3 picolyl azide.
   2. Wash wells five times with 200 µL of TBST, with shaking, 3 min per wash.
   3. Quench endogenous peroxidases with 150 µL of 0.3% H₂O₂ for 20 min at room temperature.
   4. Remove H₂O₂ and wash five times with 200 µL of TBST, with shaking 3 min per wash.
   5. Dilute streptavidin-horseradish peroxidase (SA-HRP; 1:200) in TBST and add 50 µL to each well. Incubate 1 h at room temperature.
   6. Wash five times with 200 µL of TBST, with shaking, 3 min per wash.
   7. Add 50 µL of chemiluminescent enzyme-linked immunosorbent assay (ELISA) substrate (Table of Materials) to each well.
   8. Read immediately in plate reader capable of detecting luminescence.

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

List of materials used in this article

Name	Company	Catalog Number	Comments
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