A technique used in cell culture studies of Cancer therapy

1. PROPIDIUM IODIDE
EXCLUSION ASSAY
A rapid and reliable method to quantify
viable cells in a suspension

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

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:

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

13. PRINCIPLE
 Dye binds to intracellular proteins of leaky cells.

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.

16. ADVANTAGES:
 Viable cells can be counted
 Quick and inexpensive.
 Small sample size is required.

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.