CEN Singlets

BioSure^® CEN Singlets are a 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, from human to plant. CEN Singlets are isolated from fresh chicken blood and fixed in ethanol-PBS. The procedure yields single nuclei. DNA histograms of propidium iodide (PI) stained nuclei exhibit a single peak and a small doublet peak (< 3%). The CV for the single nuclei peak is less than or equal to 3%. The published genome size is 2.5 picograms¹.

FORM

BioSure^® CEN Singlets are supplied as an unstained suspension in 2.0 milliliter volumes. The nuclei count per milliliter is at least 2.0 X 10⁷.

STABILITY

CEN Singlets 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 CEN Singlets in the PI stain prepared previously. This provides an adequate density of nuclei and sufficient working solution for several hours.

Typical CEN Singlet graph

1. Gently mix the CEN Singlet 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.