Assessment of Cell Viability

Simon Johnson1, Vy Nguyen1, David Coder2

1 University of Washington School of Medicine, Seattle, Washington, 2 Baybioscience, Irvine, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.2
DOI:  10.1002/0471142956.cy0902s64
Online Posting Date:  April, 2013
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Abstract

Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes or the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre‐fixation viability of fixed cells either before or after fixation with amine‐reactive dyes suitable for a range of excitation wavelengths. Membrane‐impermeable dead cell and live cell dyes as well as dye‐exclusion procedures for microscopy are also included. Curr. Protoc. Cytom. 64:9.2.1‐9.2.26. © 2013 by John Wiley & Sons, Inc.

Keywords: cytometry; flow cytometry; imaging; probes; toxicology; assessment of cell toxicity

     
 
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Table of Contents

  • Introduction
  • Assessment of Cell Viability Using Probes for Membrane Integrity
  • Basic Protocol 1: Propidium Iodide Staining of Nonviable Cells
  • Alternate Protocol 1: 7‐AAD Staining of Nonviable Cells
  • Alternate Protocol 2: Use of PI or 7‐AAD for Cells Labeled with PE‐Conjugated Antibodies
  • Assessment of Cell Viability Using Probes OF Physiological State
  • Alternate Protocol 3: Fluorescein Diacetate Staining of Viable Cells
  • Alternate Protocol 4: Rhodamine 123 Staining of Viable Cells
  • Assessment of Cell Viability in Fixed Cells
  • Basic Protocol 2: Live/Dead Far Red Fixable Staining of Cells for Assessment of Viability
  • Basic Protocol 3: Violet‐Excited Fixable Staining of Cells for Assessment of Viability
  • Alternate Protocol 5: Carboxymethylfluorescein Diacetate (Cell Tracker CMFDA) Staining of Cells for Assessment of Viability by Flow Cytometry
  • Alternate Protocol 6: Ethidium Monoazide Staining of Nonviable Cells Prior to Fixation
  • Alternate Protocol 7: LDS‐751 Staining of Previously Nonviable Cells After Fixation
  • Assessment of Cell Viability by Microscopy
  • Basic Protocol 4: Carboxymethyl Fluorescein Diacetate (Cell Tracker CMFDA) Staining of Cells for Assessment of Viability by Microscopy
  • Alternate Protocol 8: Simultaneous Detection of Live and Dead Cells by Microscopy using Calcein AM, EthD‐1, and Hoechst 33342
  • Alternate Protocol 9: Simultaneous Detection of Live and Dead Cells by Microscopy using Chloromethylaminocoumarin (Cell Tracker Blue CMAC) and Sytox AADvanced
  • Alternate Protocol 10: Trypan Blue Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Propidium Iodide Staining of Nonviable Cells

  Materials
  • 2 mg/ml propidium iodide (PI) in PBS (store wrapped in foil ≤1 month at 4°C)
  • Cell suspension
  • PBS ( appendix 2A)
  • 13 × 100–mm polystyrene culture tubes
CAUTION: Propidium iodide is a suspected carcinogen and should be handled with care. In particular, be careful of particulate dust when weighing out the dye. Use gloves when handling it.

Alternate Protocol 1: 7‐AAD Staining of Nonviable Cells

  • 1 mg/ml 7‐amino actinomycin D (7‐AAD; see recipe)

Alternate Protocol 2: Use of PI or 7‐AAD for Cells Labeled with PE‐Conjugated Antibodies

  • PE‐labeled cell suspension (unit 6.2)

Alternate Protocol 3: Fluorescein Diacetate Staining of Viable Cells

  • 1 mg/ml fluorescein diacetate (FDA; prepare fresh in acetone in a 13‐mm glass culture tube and cover with foil)
  • Cell suspension in culture medium appropriate for the cell type

Alternate Protocol 4: Rhodamine 123 Staining of Viable Cells

  • 1 mg/ml rhodamine 123 (prepare fresh in distilled water)
  • Cell suspension in culture medium appropriate for the cell type

Basic Protocol 2: Live/Dead Far Red Fixable Staining of Cells for Assessment of Viability

  Materials
  • Live/Dead Far Red Dead stock solution (see recipe)
  • Cell suspension, washed
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 100 mM digitonin (see recipe; optional/recommended).
  • 37% formaldehyde (Sigma, cat. no. 252549; optional)
  • PBS ( appendix 2A) with 1% (w/v) bovine serum albumin (BSA) or cell culture medium without phenol red supplemented with 1% BSA
  • 13 × 100–mm culture tubes or any suitable container for delivering cells to the flow cytometer for analysis
  • Tabletop centrifuge
  • Flow cytometer

Basic Protocol 3: Violet‐Excited Fixable Staining of Cells for Assessment of Viability

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A), azide‐free and serum/protein‐free
  • Cell suspension (∼1 × 106 cells/ml)
  • 100 mM digitonin (see recipe; optional/recommended).
  • Fixable Viability Dye eFluor 506 (eBioscience; supplied as a pre‐diluted solution in anhydrous DMSO; protect from light and moisture; store at ≤−70°C with desiccant; may be freeze‐thawed up to 20 times)
  • 37% formaldehyde (Sigma, cat. no. 252549; optional)
  • PBS ( appendix 2A) with 1% (w/v) bovine serum albumin (BSA) or cell culture medium without phenol red supplemented with 1% BSA
  • Culture tubes (13 × 100–mm) or any alternative suitable container for delivering cells to the flow cytometer for analysis
  • Flow cytometer

Alternate Protocol 5: Carboxymethylfluorescein Diacetate (Cell Tracker CMFDA) Staining of Cells for Assessment of Viability by Flow Cytometry

  • 5 mM carboxymethylfluorescein diacetate (see recipe)

Alternate Protocol 6: Ethidium Monoazide Staining of Nonviable Cells Prior to Fixation

  • 50 µg/ml ethidium monoazide (EMA; see recipe)
  • 1% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS; store mixture ≤1 week at 4°C and discard if precipitate forms)
  • 40‐W fluorescent light

Alternate Protocol 7: LDS‐751 Staining of Previously Nonviable Cells After Fixation

  • 1% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS; store mixture ≤1 week at 4°C and discard if precipitate forms)
  • 2 µg/ml LDS‐751 (laser dye styryl‐751) working solution (see recipe)

Basic Protocol 4: Carboxymethyl Fluorescein Diacetate (Cell Tracker CMFDA) Staining of Cells for Assessment of Viability by Microscopy

  Materials
  • 5 mM carboxymethylfluorescein diacetate (see recipe)
  • 1 mM Hoechst 33342 (Sigma) in phosphate‐buffered saline (PBS; appendix 2A)
  • Cell culture medium (prewarmed to 37°C)
  • Cultured cells on chambered coverglass (Nunc, cat. no. 155379) or grown on coverslips (for fixed only)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fluorescence microscope with filters appropriate for FITC and DAPI

Alternate Protocol 8: Simultaneous Detection of Live and Dead Cells by Microscopy using Calcein AM, EthD‐1, and Hoechst 33342

  Materials
  • 2 mM Calcein AM (see recipe)
  • 4 mM ethidium homodimer 1 (EtHD‐1; see recipe)
  • Cell culture medium (prewarmed to 37°C)
  • Cultured cells on chambered covers glass (Nunc, cat. no. 155379) or grown on coverslips (for fixed cells only)
  • 100 mM digitonin (see recipe) or 50% (v/v) Triton X‐100 in H 2O; optional/recommended
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fluorescence microscope with filters for FITC (for Calcein AM), Texas Red (for EthD‐1), and DAPI (for Hoechst)

Alternate Protocol 9: Simultaneous Detection of Live and Dead Cells by Microscopy using Chloromethylaminocoumarin (Cell Tracker Blue CMAC) and Sytox AADvanced

  Materials
  • 1 mM CMAC (see recipe)
  • 1 mM Sytox AADvanced (see recipe)
  • Cell culture medium (prewarmed to 37°C)
  • Cultured cells on chambered covers glass (Nunc, cat. no. 155379) or grown on coverslips (for fixed cells only)
  • 100 mM digitonin (see recipe) or 50% (v/v) Triton X‐100 in H 2O; optional/recommended
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fluorescence microscope with filters appropriate for 353 nm absorption/466 nm emission (CMAC) and 546 nm absorption/647 emission (for Sytox AADvanced)

Alternate Protocol 10: Trypan Blue Staining

  • 0.4% (w/v) trypan blue in PBS (store up to 1 year at room temperature in the dark; filter if a precipitate forms; for PBS, see appendix 2A)
  • Serum‐free culture medium ( appendix 3B optional)
  • Additional materials for cell counting with a hemacytometer ( appendix 3A)
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Figures

  •   FigureFigure 9.2.1 Identification of nonviable cells with propidium iodide (PI). Nonviable cells are more than two decades brighter than the unstained, viable cells. Gating on a one‐parameter histogram is sufficient to identify the viable population. Region 1: viable cells; region 2: nonviable cells.
  •   FigureFigure 9.2.2 Effects of gating and compensation with 7‐AAD. (A) Gating discriminates live cells. One‐parameter histogram of logarithmically amplified 7‐AAD fluorescence using a 650‐nm long‐pass filter. Mouse spleen cells are labeled only with 7‐AAD. Note the peak of dead cell population in region 2 at a relative brightness between 100 and 200 (about ten‐fold dimmer than what is expected for propidium iodide). The live cell population that occupies the first decade in the histogram (region 1) is 7‐AAD negative and constitutes the majority of the cells in the population. (B) Uncompensated phycoerythrin fluorescence in the presence of 7‐AAD. A bivariate plot of mouse spleen cells labeled with 7‐AAD and a PE‐labeled antibody to a cell surface antigen. Note the two small populations of dead cells in regions 2 and 4 and the large population of live cells that occupies region 2. If gating was performed before adequate compensation was achieved, then most of the viable PE‐positive cells could be lost. (C) Compensation of PE with 7‐AAD. Distribution of cells from same sample as in B. Note the dead cells are in the same location as in B, but the live cells are now clearly resolved from 7‐AAD positive populations. At this point, the live cell gate defined in region 2 of A is valid.
  •   FigureFigure 9.2.3 Effects of gating with rhodamine 123. (A) Identification of live cells after gating. Rhodamine 123 may not always completely resolve viable from nonviable cells as indicated in the un‐gated histogram (dotted line). Gating on forward light scatter versus rhodamine 123 fluorescence helps separate both populations. Note the histogram of the gated population of viable cells (solid line) overlaid on the ungated population. (B) A bivariate plot of forward light scatter versus rhodamine 123 fluorescence helps to resolve live (rhodamine 123–bright) and dead (rhodamine 123–dim) populations. Debris is gated out at the same time.
  •   FigureFigure 9.2.4 Example of staining and flow cytometry analysis of live/dead populations using fixed cells stained that had been stained with Live/Dead Deep Red. (A) Sample plot of a confluent population of cells. Live cells stain weakly, while dead cells, in this case representing 17% of the culture population, stain brightly. (B) A positive control for dead cells produced by treating cells with 100 µM digitonin during staining. (C) An overlay of the sample, dead cell control, and an intermediate concentration of digitonin demonstrating that partial staining may occur if cells are partially permeabilized. This was only observed when cells were permeabilized using digitonin and does not occur in normal cell populations.
  •   FigureFigure 9.2.5 Sample staining using Cell Tracker CMFDA. Stained dead cells are brighter than unstained cells while stained live cells are >100 fold brighter than dead cells and easily distinguished from the dead population.
  •   FigureFigure 9.2.6 Identification of dead cells with eFluor 506. The bright dead population is clearly distinguished from unstained cells. While the excitation was with 405‐nm light, the detection filter, (530/30), was slightly suboptimal for detection.
  •   FigureFigure 9.2.7 Example of viability analysis using Cell Tracker CMFDA and microscopy. (A) Cells are efficiently permeabilized using Triton X‐100 as a positive control and CMFDA shows a steep dose response curve indicating high specificity for permeabilized cells. (B) Live cells are extremely bright and easily distinguished from dead cells. A counterstain is necessary to present data as a percent of total.
  •   FigureFigure 9.2.8 Example of dual staining with Calcein AM and EthD‐1. (A) Analysis of dual‐stained cells permeabilized with Triton X‐100 demonstrates that while both dyes are highly specific for their target cells (they have steep drop‐offs of staining in the dose response curve), they are slightly non‐overlapping in their staining. Calcein AM is lost before EthD‐1 stains cells (see inlay). This analysis did not include weakly staining cells, and adjustment of brightness to include these cells may reduce the observed lack of complementation. (B) Cells stained with either dye are extremely bright and easy to distinguish from unstained cells, although intermediate permeabilization can result in a lack of either stain.
  •   FigureFigure 9.2.9 Sample staining using Cell Tracker Blue CMAC and Sytox AADvanced. (A) Dose response curve for staining and permeabilization with Sytox AADvanced and CMAC. (B) These dyes appeared to be highly complementary, with no apparent intermediate dose where cells lacked staining for either dye, although no total cell staining dye was included in this analysis.

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Literature Cited

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