ZebraFish nuclei, the newest addition to the BioSure®
DNA controls and standards, are produced from ZebraFish (Danio rerio
), with a genome size of 3.4 - 3.5 pg. Use this product with TENs (rainbow trout) and TTNs (triploid rainbow trout) to provide you with a triple set of flow cytometry reference points.
BioSure® ZebraFish Nuclei (ZFNs) are an internal biological reference material that can function as a DNA standard or control when determining nuclear DNA content by flow cytometry. The DNA content of ZebraFish red blood cell nuclei has been reported to be 3.4 - 3.5 picograms1. These fish are also known as Danio rerio and Zebra danio.
ZFNs are isolated from whole blood and fixed in ethanol-PBS. The procedure yields single nuclei and a few doublets and larger aggregates. DNA linear scale histograms of propidium iodide (PI) stained nuclei exhibit multiple peaks; single nuclei constitute peak one. Other performance characteristics for this product have not been established.
BioSure® ZFNs are supplied as an unstained suspension in 2.0 milliliter volumes. The nuclei count per milliliter is at least 2.0 X 105.
ZFNs 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. Or, purchase BioSure®
PI Staining Solution, Catalog No. 1021.
PREPARATION OF WORKING SOLUTION
The recommended working solution is a 1:10 dilution of the stock ZFNs in the PI stain, 50 µg/mL (PI Stain Catalog No. 1021). This provides an adequate density of nuclei and sufficient working solution for several hours.
Typical ZebraFish graph
1. Gently mix the ZFNs stock for 5 - 10 seconds to suspend the nuclei and add two drops to a test tube (13 X 75 mm).
2. Add 1.0 mL of PI stain (Cat. No. 1021), 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 4 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.