( l lysate and vortexed. This subsequent ...)Improving the accuracy and speed of mammalian cell counting

l lysate and vortexed. This subsequent cell lysate is loaded into a Nucleo- Cassette(New Brunswick Scientific Co., Inc., figure 4), where the nuclei are stained with PI, by immersing the tip of the cassette into the sample mixture and depressing the loading piston; this loads a predefined volume (~50 μL) of the sample mixture into the cassette. The Nucleo- Cassette is then placed into the NucleoCounter for analysis. The lysate dissolves the Propidium Iodide, which is immobilized in the cassette, and a mechanical drive in the instrument then transfers the sample into the measuring area of the cassette (figure 4). During analysis the fluorescent signal is registered and correlated to a total cell count (figure 5). The total cell concentration in the NucleoCassette is presented in the NucleoCounter display as cells per milliliter. Optionally, the data can be presented on a PC using the NucleoView software (New Brunswick Scientific Co., Inc.).

Determination of viability

The total cell determination is achieved by deliberately disrupting the plasma membranes of all cells in a sample by pretreatment with reagent A-100 and reagent B. All nuclei are therefore accessible to PI staining, independent of whether cells initially were viable or nonviable prior to cell lysis. However, by also measuring a cell sample without pretreatment, (no addition of Reagent A- 100 or B), only cells with pre-impaired plasma membranes are PI-stained (figure 6). Analysis in the system then gives an estimate of the concentration of nonviable cells in the original cell suspension. By using these two measurements, the viability can be automatically calculated and displayed via the NucleoView software. Alternately, a manual calculation can be applied.

where V=Viability; C=Cell concentration; M=Multiplication (dilution) factor; t=Total cell count; nv=Non-viable cell count.

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