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Protocol for Live/Dead Count - University of Florida

Differential Staining of Live and Dead Embryos using Fluorescein Diacetate

and Ethidium Bromide

J. Lannett Edwards and P.J. Hansen

Dept. of Animal Sciences, University of Florida

(JLE is now at University of Tennessee)

List of Other Protocols  P.J. Hansen Home Page   |  Dept .of Animal Sciences


University of Florida


 

Procedures obtained from Ann Croy (Guelph, Canada) as a modification of procedures detailed in Transplantation 1971 12:148-151 Takasugi, M. An improved fluorochromatic cytotoxic test. Note that the procedure can be used for staining of all cells and not just embryos or oocytes. Also, ethididium bromide can be replaced with propidium iodide, DAPI or Hoescht 33342

 

Materials

 

Ethidium Bromide (EtBr; Fisher)

Fluorescein Diacetate (FDA; Sigma)

DPBS

Acetone

 

Procedure

1) Make following stock solutions:

 

EtBr (10 mg/mL DPBS) Store in dark at 4 C

FDA (5 mg/mL acetone) Store in dark in glass container at -20 C

 

Storage life of stocks ~4 months

 

2) Just before use (i.e., ~10 min) prepare the following in a 15 mL conical tube covered with aluminum foil:

 

100 μl EtBr Stock (0.05 mg/mL)

3 μl FDA Stock (0.005 mg/mL)

10 mL DPBS or culture medium

Note: If you want to recover embryos from glass slide, use DPBS with 0.1% BSA or serum to prevent them from sticking to slide.

 

3) Place on ice until needed.

 

4) To stain embryos or oocytes place 50 μl of dye solution on a glass slide.

 

5) In the smallest volume possible transfer embryos or oocytes to be stained in the 50 μl and allow to sit in the dark for at least 3 min (FDA cleavage of acetate radical traps dye inside cell; 3 min=time for accumulation).

6) View embryos or oocytes for staining using the fluorescence microscope under UV epiluminesence (use UV filter).

 

Live Stain=Green

Dead Stain=Red/Orange

 

Count green first before it "burns out" from illumination

 

 


       Page maintained by P.J. Hansen
       hansen@animal.ufl.edu
       http://www.animal.ufl.edu/hansen
       Protocol created:
February 2, 1995; online 02-21-2005
       Last updated: February 22, 2005

       Original material J. Lannett Edwards and Peter J. Hansen

       Links to commercial sites do not constitute endorsement by the

authors or the University of Florida.  

 University of Florida
Department of Animal Sciences
PO Box 110910
Gainesville, Florida 32611
Phone:(352)
392-5590
Fax:(352)
392-3922