2. Determination of cell viability is critical when evaluating
the response to cytotoxic drugs or other environmental
factors.
In addition, it is often necessary to detect dead cells in a
cell suspension in order to exclude them from the
analysis.
Dead cells can generate artifacts as a result of
nonspecific antibody binding or through unwanted uptake
of fluorescent probes.
PURPOSE:
3. PROPIDIUM IODIDE
PI -- intercalating agent
fluorescent molecule
Molecular mass -- 668.4 Da
Can be used to stain cells
4. PI is a membrane impermeant dye -- generally excluded
from viable cells
Penetrate cell membranes of dying or dead cells
Binds to double stranded DNA by intercalating between
base pairs
When PI does gain access to nucleic acids and
intercalates -- fluorescence increases dramatically --
therefore used to identify dead cells
Stained cells are determined
Flow cytometery
Flourescent microscopy
5.
6.
7. FACS™ Tubes (5 mL round-bottom polystyrene tubes)
Pipette Tips and Pipettes
Centrifuge
Vortex
MATERIALS REQUIRED:
8. PBS (1X): 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4 or Hank’s
Balanced Salt Solution (HBSS; 1X)
Flow Cytometry Fixation Buffer (R&D Systems,
Catalog # FC004, or an equivalent solution containing BSA and sodium
azide)
PI Staining Solution: 10 μg/ml PI in PBS stored at 4 °C in
the dark
Detection Antibodies (optional)
Isotype Control Antibodies (optional)
REAGENTS REQUIRED
9. Harvest cells and aliquot up to 1 x 106 cells/100 μL into FACS tubes. Wash
the cells 2 times by adding 2 mL of PBS, centrifuging at 300 x g for 5
minutes, and then decanting the buffer from the pelleted cells.
Resuspend cells in 100 μL of Flow Cytometry Staining Buffer.
Add 5 - 10 μL of PI staining solution. Mix gently and incubate for 1 minute
in the dark.
Determine PI fluorescence with a FACScan™ instrument.
Note: Do not wash cells after the addition of the PI staining solution.
PROCEDURE:
10.
11. ADVANTAGES
Quick and inexpensive
Simple, rapid and reliable method for assessing
viable cells
Only a small fraction of total cells from a population
is required
12. TRYPAN BLUE ASSAY
A rapid and reliable method to quantify viable cells in a
suspension
14. procedure:
Incubation with trypan blue dye.
Live cells can not take up because of intact
cell membranes.
Only dead cells are stained.
Visualization:
Light microscope
Automated cell counter and analyzer e.g;
Cedex XS Analyzer or Cedex HiRes
Analyzer.
17. DISADVANTAGES:
Every sample must be counted individually.
Subjective evaluation with hemocytometer is a
limitation.
Not detection of apoptosis.
Detects only necrotic cells.
18. LIMITATIONS
Each individual sample must be counted; only a few
tests may be simultaneously performed.
Not specific for apoptosis
PI is a suspected carcinogen and should be
handled with care. The dye must be disposed of
safely and in accordance with applicable local
regulations.