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Assessment of Cell Viability

David M. Coder1

1University of Washington School of Medicine, Seattle, Washington

Publication Name: 
Current Protocols in Cytometry
Unit Number: 
Unit 9.2
DOI: 
10.1002/0471142956.cy0902s15
Online Posting Date: 
May, 2001
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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 of 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. A dye-exclusion procedure for microscopy is also included.

     
 
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Table of Contents

  • Unit Introduction
  • Assessment of Cell Viability Using Probes for Membrane Integrity
  • Basic Protocol: 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
  • Alternate Protocol 5: Ethidium Monoazide Staining of Nonviable Cells Prior to Fixation
  • Alternate Protocol 6: LDS-751 Staining of Previously Nonviable Cells After Fixation
  • Assessment of Cell Viability by Microscopy
  • Alternate Protocol 7: Using Trypan Blue Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol:  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
 Additional Materials (also see Basic Protocol)
  • 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
 Additional Materials (also see Basic Protocol)
  • PE-labeled cell suspension (unit 6.2)
Alternate Protocol 3:  Fluorescein Diacetate Staining of Viable Cells
 Additional Materials (also see Basic Protocol)
  • 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
 Additional Materials (also see Basic Protocol)
  • 1 mg/ml rhodamine 123 (prepare fresh in distilled water)
  • Cell suspension in culture medium appropriate for the cell type
Alternate Protocol 5:  Ethidium Monoazide Staining of Nonviable Cells Prior to Fixation
 Additional Materials (also see Basic Protocol)
  • 50 µg/ml ethidium monoazide (EMA; see recipe)
  • 1% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS recipe; store mixture £1 week at 4°C and discard if precipitate forms)
  • 40-W fluorescent light
Alternate Protocol 6:  LDS-751 Staining of Previously Nonviable Cells After Fixation
 Additional Materials (also see Basic Protocol)
  • 1% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS recipe; 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)
Alternate Protocol 7:  Using Trypan Blue Staining
 Additional Materials (also see Basic Protocol)
  • 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 recipe, 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

  • Figure 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.

    View Image
  • Figure 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.

    View Image
  • Figure 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 ungated 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.

    View Image

Literature Cited

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