BioSure® Triploid Trout Erythrocyte Nuclei are a biological reference material that can function as a DNA standard when determining nuclear DNA content of human, animal, or plant cells by flow cytometry1. The triploid rainbow trout is an artificially produced polyploid. Triploid nuclei are isolated from fresh rainbow (3N) trout blood and fixed in ethanol-PBS. The published genome size is 7.8 picograms2. The procedure yields single nuclei and a few larger aggregates. Other performance characteristics for this product have not been established.
FORM
BioSure® Triploid Nuclei are supplied as an unstained suspension in 2.0 milliliter volumes. The nuclei count per milliliter is at least 2.0 X 107.
STABILITY
Triploid Nuclei are stable at 2° - 8° C until the expiration date denoted on each vial label.
PREPARATION OF PROPIDIUM IODIDE (50 microgram/mL) STAINING SOLUTION
To 48 milliliters of calcium and magnesium free Dulbecco's PBS, add 2.5 mg of propidium iodide (PI) and 0.3 mL of Nonidet P40 (NP40). Mix the solution and Q.S. to 50 mL. Staining solution is stable at 2° - 8° C for at least 30 days when stored protected from light in an amber bottle.
PREPARATION OF PROPIDIUM IODIDE (50 microgram/mL) STAINING SOLUTION
To 48 milliliters of calcium and magnesium free Dulbecco's PBS, add 2.5 mg of propidium iodide (PI) and 0.3 mL of Nonidet P40 (NP40). Mix the solution and Q.S. to 50 mL. Staining solution is stable at 2° - 8° C for at least 30 days when stored protected from light in an amber bottle.
PREPARATION OF WORKING SOLUTION
The recommended working solution is a 1:10 dilution of the stock Triploid Nuclei in the PI stain prepared previously. This provides an adequate density of nuclei and sufficient working solution for several hours.
Typical Triploid Trout graph
1. Gently mix the Triploid Nuclei stock for 5 - 10 seconds to suspend the nuclei, and add one or two drops to a test tube (13 X 75 mm).
2. Add 1.0 mL of PI stain, mix for 5 - 10 seconds, and incubate at room temperature (22° - 25° C) for ten minutes protected from light.
3. Mix sample 5 - 10 seconds and evaluate. Samples are typically stable at 2° - 8° C for four hours when protected from light.
NOTE: The flow cell of some cytometers is susceptible to clogging. Filtering the stained preparation through a 30 - 50 mesh nylon screen will eliminate large nuclear aggregates.
LITERATURE CITED
1Pierrez, J., Ronot, X.: Use of Diploid and Triploid Trout Erythrocytes as Internal Standards in Flow Cytometry. Cytometry 12: 275-278, 1991.
2Gregory, T.R.; Animal Genome Size Database: 2001-2005, available at http://www.genomesize.com.
