Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry

Scott T. Clarke1, Veronica Calderon1, Jolene A. Bradford1

1 Thermo Fisher Scientific, Eugene
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.49
DOI:  10.1002/cpcy.24
Online Posting Date:  October, 2017
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Abstract

The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S‐phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis. This chapter presents a pulse labeling method using the thymidine analog, 5‐ethynyl‐2′‐deoxyuridine (EdU), with subsequent detection by click chemistry. EdU detection using click chemistry is bio‐orthogonal to most living systems and does not non‐specifically label other biomolecules. Live cells are first pulsed with EdU. After antibody labeling cell surface markers, fixation, and permeabilization, the incorporated EdU is covalently labeled using click chemistry thereby identifying proliferating cells. Improvements in click chemistry allow for labeling in the presence of fluorescent proteins and phycobiliproteins without quenching due to copper. Measuring DNA replication during cell cycle progression has cell health applications in flow cytometry, fluorescence microscopy, and high content imaging. This protocol has been developed and optimized for research use only and is not suitable for use in diagnostic procedures. © 2017 by John Wiley & Sons, Inc.

Keywords: EdU; S‐phase; click chemistry; proliferation; thymidine analog; picolyl azide; cell cycle; GFP compatibility; R‐PE compatibility

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Flow Cytometric Analysis of EdU Combined with DNA Content
  • Basic Protocol 2: Flow Cytometric Analysis of EdU, Combined with DNA Content and Antibody Staining for Cell Surface Markers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Flow Cytometric Analysis of EdU Combined with DNA Content

  Materials
  • Cells cultured in appropriate culture medium
  • Click‐iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific; see recipe)
  • Trypsin/EDTA (see recipe)
  • Phosphate‐buffered saline (PBS), pH 7.2‐7.4 (see recipe)
  • Antibody diluting buffer (see recipe)
  • FxCycle Violet stain (DAPI; see recipe)
  • 37°C incubator
  • 5‐ml, 12 × 75–mm polystyrene tubes
  • 15‐ml screw‐capped centrifuge tubes
  • 6‐well cell culture dish or 75‐cm2 cell culture flask
  • Benchtop centrifuge
  • Device for counting cells, such as a hemacytometer or Countess automated cell counter (Invitrogen, cat. no. C10227)
  • Multi‐laser flow cytometer equipped with 405 nm and 633/640 nm excitation lasers (e.g., Invitrogen Attune NxT Flow Cytometer or Becton Dickenson BD LSR II): For detection of green fluorescent protein (GFP) the addition of the 488‐nm excitation laser will be required, and the addition of other fluorescent proteins may require the addition of alternate excitation laser lines (e.g., 561‐nm laser excitation for mCherry fluorescent protein)
NOTE: Handling and Disposal: EdU are nucleoside analogs which can be incorporated into DNA. Handle and dispose of each in compliance with all pertaining local regulations. When dissolved in DMSO, which is known to facilitate the entry of organic molecules into tissue, use additional precautions appropriate for the hazards posed by such materials.CAUTION: The hazards posed by FxCycle Violet stain have not been fully investigated. The stain is a known mutagen and may cause sensitization by inhalation and skin contact, and is irritating to eyes, respiratory system, and skin. Do not breathe dust. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing, safety glasses, and gloves. Avoid contact with skin and eyes. Dispose of the reagents in compliance with all pertaining local regulations.CAUTION: DMSO, provided as a solvent, is known to facilitate the entry of organic molecules into tissues. Handle reagents containing DMSO using practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with all pertaining local regulations.

Alternate Protocol 1:

  Additional Materials (also see protocol 1)
  • Click‐iT Plus EdU Alexa Fluor 350 Flow Cytometry Assay Kit (Invitrogen): The entire kit is stored at 2° to 6°C and is stable for up to one year (this kit differs from the other kits only in the color of the azide component; refer to Table 7.49.2 for additional EdU dye choices)
  • SYTOX AADvanced stain (see recipe)
  • RNase A, 20 mg/ml (Invitrogen, cat. no. 12091‐039)
  • Multi‐laser flow cytometer equipped with 355‐nm and 488‐nm excitation lasers (e.g., Becton Dickenson BD LSR II): For detection of green fluorescent protein (GFP), the addition a 530/30‐nm bandpass filter with the 488‐nm excitation laser will be required, and the addition of other fluorescent proteins may require the addition of alternate excitation laser lines (e.g., 561‐nm laser excitation for mCherry fluorescent protein)
Table 7.9.2   Additional Materials (also see protocol 1)Click‐iT Plus EdU Flow Cytometry KitsDNA Binding Dyes Used for DNA Content Measurement by Flow Cytometry, with Excitation and Emission Information

EdU fluorophore Excitation maximum (λ) in nm Laser line (λ) in nm Emission maximum (λ) in nm
Alexa Fluor 350 picolyl azide a 350 355 438
Pacific Blue picolyl azide a 410 405 455
Alexa Fluor 488 picolyl azide a 495 488 519
Alexa Fluor 594 picolyl azide a 590 532, 561 617
Alexa Fluor 647 picolyl azide a 650 633, 640 668
DNA‐binding dyes for DNA content in fixed cells Excitation maximum (λ) in nm Laser line (λ) in nm Emission maximum (λ) in nm Requires use of RNase
7‐aminoactinomycin‐D (7‐AAD) a 546 488, 532, 561 647 No
Propidium iodide a 535 488, 532, 561 617 Yes
4′,6‐diamidino‐2‐phenylindole (DAPI) a 350 355, 405 470 No
FxCycle Far Red b 640 633, 640 658 Yes
FxCycle Violet b 350 355, 405 470 No
FxCycle PI/RNase b 535 488, 532, 561 617 No c
FxCycle Violet Ready Flow b 350 355, 405 470 No
SYTOX AADvanced b 546 488, 532, 561 647 Yes
SYTOX Blue b 445 405 470 Yes
SYTOX Green b 504 488 523 Yes
SYTOX Orange b 547 488, 532, 561 570 Yes

 aThermo Fisher Scientific (all names are trademarked)
CAUTION: The hazards posed by all of the nucleic acid binding stains (listed in Table 7.49.3) have not been fully investigated. Since the stain is known to bind to nucleic acids, treat the stain as a potential mutagen and use with appropriate care. The nucleic acid binding stains are used as a solution in dimethyl sulfoxide (DMSO), which is known to facilitate the entry of organic molecules into tissues. Handle the stain in DMSO using equipment and practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with all pertaining local regulations.
Table 7.9.3   Additional Materials (also see protocol 1)Click‐iT Plus EdU Flow Cytometry KitsDNA Binding Dyes Used for DNA Content Measurement by Flow Cytometry, with Excitation and Emission Information

EdU fluorophore Excitation maximum (λ) in nm Laser line (λ) in nm Emission maximum (λ) in nm
Alexa Fluor 350 picolyl azide a 350 355 438
Pacific Blue picolyl azide a 410 405 455
Alexa Fluor 488 picolyl azide a 495 488 519
Alexa Fluor 594 picolyl azide a 590 532, 561 617
Alexa Fluor 647 picolyl azide a 650 633, 640 668
DNA‐binding dyes for DNA content in fixed cells Excitation maximum (λ) in nm Laser line (λ) in nm Emission maximum (λ) in nm Requires use of RNase
7‐aminoactinomycin‐D (7‐AAD) a 546 488, 532, 561 647 No
Propidium iodide a 535 488, 532, 561 617 Yes
4′,6‐diamidino‐2‐phenylindole (DAPI) a 350 355, 405 470 No
FxCycle Far Red b 640 633, 640 658 Yes
FxCycle Violet b 350 355, 405 470 No
FxCycle PI/RNase b 535 488, 532, 561 617 No c
FxCycle Violet Ready Flow b 350 355, 405 470 No
SYTOX AADvanced b 546 488, 532, 561 647 Yes
SYTOX Blue b 445 405 470 Yes
SYTOX Green b 504 488 523 Yes
SYTOX Orange b 547 488, 532, 561 570 Yes

 aMultiple manufacturers.
 bThermo Fisher Scientific (all names are trademarked).
 cRNase is added into the formulation.

Alternate Protocol 2:

  Additional Materials (also see protocol 1)
  • Click‐iT Plus EdU Alexa Fluor 350 Flow Cytometry Assay Kit (Invitrogen): The entire kit is stored at 2° to 6°C and is stable for up to one year (this kit differs from the other kits only in the color of the azide component)
  • FxCycle PI/RNase stain (Invitrogen, cat. no. F10797): For long‐term storage, the dye is supplied as a 1× room temperature stable solution stable for at least 1 year (This ready‐to‐use formulation contains optimal amount of RNase in the PI solution)
  • Multi‐laser flow cytometer equipped with 355‐nm and 488‐, 532‐, or 561‐nm excitation lasers (e.g., Becton Dickenson BD LSR II): For detection of green fluorescent protein (GFP) the addition a 530/30 nm bandpass filter with the 488‐nm excitation laser will be required,, and the addition of other fluorescent proteins may require the addition of alternate excitation laser lines (e.g., 561‐nm laser excitation for mCherry fluorescent protein)
CAUTION: The hazards posed by the FxCycle PI/RNase stain have not been fully investigated. The solution contains propidium iodide, which binds to nucleic acids and should be treated as a potential mutagen. Dispose of the reagents in compliance with all pertaining local regulations.

Alternate Protocol 3:

  Additional Materials (also see protocol 1)
  • Click‐iT Plus EdU Pacific Blue Flow Cytometry Assay Kit (Invitrogen): The entire kit is stored at 2° to 6°C and is stable for up to one year (this kit differs from the other kits only in the color of the azide component)
  • SYTOX AADvanced stain (see recipe)
  • RNase A, 20 mg/ml (Invitrogen, cat. no. 12091‐039) Multi‐laser flow cytometer equipped with 405‐nm and 488‐nm excitation lasers (e.g., Invitrogen Attune NxT Flow Cytometer or Becton Dickenson BD LSR II): For detection of green fluorescent protein (GFP), the addition a 530/30 nm bandpass filter with the 488‐nm excitation laser will be required, and the addition of other fluorescent proteins may require the addition of alternate excitation laser lines (e.g., 561‐nm laser excitation for mCherry fluorescent protein)
CAUTION: The hazards posed by the SYTOX AADvanced stain have not been fully investigated. Since the stain is known to bind to nucleic acids, treat the stain as a potential mutagen and use with appropriate care. The SYTOX AADvanced stain is used as a solution in DMSO, which is known to facilitate the entry of organic molecules into tissues. Handle the stain in DMSO using equipment and practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with all pertaining local regulations.

Alternate Protocol 4:

  Additional Materials (also see protocol 1)
  • Click‐iT Plus EdU Pacific Blue Flow Cytometry Assay Kit (Invitrogen): The entire kit is stored at 2° to 6°C and is stable for up to one year (this kit differs from the other kits only in the color of the azide component)
  • FxCycle PI/RNase stain (Invitrogen cat. no. F10797): For long‐term storage, the dye is supplied as a 1× room temperature stable solution stable for at least 1 year (this ready‐to‐use formulation contains optimal amount of RNase in the PI solution)
  • Multi‐laser flow cytometer equipped with 405‐nm excitation (laser) for detection of Pacific Blue picolyl azide and 488‐, 532‐, or 561‐nm excitation lasers for detection of propidium iodide (e.g., Invitrogen Attune NxT Flow Cytometer or Becton Dickenson BD LSR II): For detection of green fluorescent protein (GFP), the addition of a 530/30 nm bandpass filter with the 488‐nm excitation laser will be required, and the addition of other fluorescent proteins may require the addition of alternate excitation laser lines (e.g., 561‐nm laser excitation for mCherry fluorescent protein)
CAUTION: The hazards posed by the FxCycle PI/RNase stain have not been fully investigated. The solution contains propidium iodide, which binds to nucleic acids and should be treated as a potential mutagen. Dispose of the reagents in compliance with all pertaining local regulations.

Alternate Protocol 5:

  Additional Materials (also see protocol 1)
  • Click‐iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen): The entire kit is stored at 2° to 6°C and is stable for up to 1 year (this kit differs from the other kits only in the color of the azide component)
  • Multi‐laser flow cytometer equipped with 488‐nm excitation for detection of Alexa Fluor 488 picolyl azide and 405‐nm excitation lasers for detection of FxCycle Violet and 532‐nm or 561‐nm excitation laser for the detection of mCherry or dtTomato (e.g., Invitrogen Attune NxT Flow Cytometer or Becton Dickenson BD LSR II)

Alternate Protocol 6:

  Additional Materials (also see protocol 1)
  • Click‐iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen): The entire kit is stored at 2° to 6°C and is stable for up to one year (this kit differs from the other kits only in the color of the azide component)
  • FxCycle Far Red stain (see recipe)
  • RNase A, 20 mg/ml (Invitrogen, cat. no. 12091‐039) Multi‐laser flow cytometer equipped with 488‐nm excitation for detection of Alexa Fluor 488 picolyl azide and 633/637‐nm excitation lasers for detection of FxCycle Far Red and 532‐nm or 561‐nm excitation laser for the detection of mCherry or dtTomato (e.g., Invitrogen Attune NxT Flow Cytometer or Becton Dickenson BD LSR II)

Alternate Protocol 7:

  Additional Materials (also see protocol 1)
  • Click‐iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay Kit (Invitrogen): The entire kit is stored at 2° to 6°C and is stable for up to one year (this kit differs from the other kits only in the color of the azide component)
  • SYTOX AADvanced stain (see recipe)
  • RNase A, 20 mg/ml (Invitrogen, cat. no. 12091‐039) Multi‐laser flow cytometer equipped with 561 nm excitation for detection of Alexa Fluor 594 picolyl azide and 488 nm excitation lasers for detection of SYTOX AADvanced. For detection of green fluorescent protein (GFP) the addition a 530/30 nm bandpass filter with the 488 nm excitation laser will be required and 532 nm or 561 nm excitation laser for the detection of mCherry or dtTomato (e.g., Invitrogen Attune NxT Flow Cytometer or Becton Dickenson BD LSR II).

Basic Protocol 2: Flow Cytometric Analysis of EdU, Combined with DNA Content and Antibody Staining for Cell Surface Markers

  Materials
  • Peripheral blood mononuclear cells (PBMCs) or cells cultured in appropriate tissue culture medium
  • Click‐iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit (see recipe)
  • Trypsin/EDTA (see recipe)
  • Phosphate‐buffered saline (PBS), pH 7.2‐7.4 (see recipe)
  • Antibody diluting buffer (see recipe)
  • Anti‐CD8 PE conjugate clone 3B5 (Invitrogen): The antibody conjugate is supplied in a solution of phosphate‐buffered saline (PBS), pH 7.2, containing 5 mM sodium azide (the conjugate is stable at 2° to 6°C for up to 1 year)
  • Anti‐CD3 PerCP conjugate clone UCHT1: The antibody conjugate is supplied in a phosphate‐buffered saline (PBS), pH 7.2, containing 14 mM sodium azide and 0.2% bovine serum albumin (BSA); the conjugate is stable at 2° to 6°C for several years.
  • FxCycle Far Red stain (see recipe)
  • RNase A (20 mg/ml; Invitrogen, cat. no. 12091‐039)
  • 37°C incubator
  • 5‐ml, 12 × 75–mm polystyrene tubes
  • 15‐ml screw‐capped centrifuge tubes
  • 6‐well cell culture dish or 75‐cm2 culture flask
  • Benchtop centrifuge
  • Device for counting cells, such as a hemacytometer or Countess automated cell counter (Invitrogen cat. no. C10227)
  • Multi‐laser flow cytometer equipped with 405‐nm, 488‐, 532‐ or 561‐, and 633/640‐nm excitation lasers (e.g., Invitrogen Attune NxT Flow Cytometer or Becton Dickenson BD LSR II)
CAUTION: Dimethyl sulfoxide (DMSO), provided as a solvent, is known to facilitate the entry of organic molecules into tissues. Handle reagents containing DMSO using practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with all pertaining local regulations.CAUTION: The hazards posed by FxCycle Far Red stain have not been fully investigated. The stain is a known mutagen and may cause sensitization by inhalation and skin contact, and is irritating to eyes, respiratory system, and skin. Do not breathe dust. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing, safety glasses, and gloves. Avoid contact with skin and eyes. Dispose of the reagents in compliance with all pertaining local regulations.NOTE: PBMCs can be treated with various stimulants [e.g., phorbol‐12‐ myristate‐13‐acetate (PMA) + ionomycin, SEB, or CD3e + CD28 monoclonal antibodies] resulting in proliferation. In this protocol, PMA plus ionomycin are used to stimulate the PBMCs. Concentration and duration of stimulant treatment needs to be optimized.
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.scripps.edu/sharpless/click.html
  Sharpless’ Web site for click chemistry.
  www.thermofisher.com/edu
  Thermo Fisher Scientific EdU click chemistry Web site.
  www.thermofisher.com/spectraviewer
  Thermo Fisher Scientific Fluorescence SpectraViewer.
  www.thermofisher.com/bioprobes
  BioProbes Journal of Cell Biology Applications.
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