BioSure® Chicken Erythrocyte Nuclei (CENs) are an internal biological reference material that can function as a performance standard when calibrating and troubleshooting flow cytometers. This control can be used as a reference for all types of cells. CENs are isolated from fresh chicken blood and fixed in ethanol-PBS. The procedure yields single nuclei, doublets, triplets and a few larger aggregates. DNA linear scale histograms of propidium iodide (PI) stained nuclei exhibit multiple peaks; single nuclei constitute peak one (nearest the ordinate) while the next three peaks to the right have mean channel values which are approximately 2, 3, and 4 times greater than the single nuclei peak. A properly aligned flow cytometer will also have a CV for the single nuclei peak of less than or equal to 3%. The published genome size is 2.5 picograms1.
BioSure® CENs are supplied as an unstained suspension in 2.0 milliliter volumes. The nuclei count per milliliter is at least 2.0 X 107.
CENs 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 WORKING SOLUTION
The recommended working solution is a 1:10 dilution of the stock CENs in the PI stain prepared previously. This provides an adequate density of nuclei and sufficient working solution for several hours.
Typical CEN graph
1. Gently mix the CEN 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 10 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.
Although the Negative Control has been thoroughly washed to remove residual fixative, the chemical is hazardous, and it is recommended that gloves and eye protection be worn when handling this product.
1 Gregory, T.R.; Animal Genome Size Database: 2001-2005, available at http://www.genomesize.com.