ChemoMetec NC-100 - Manual (Page 39)

6 Total and viability count
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6 Total and viability count
This chapter describes the basic procedures for the determination of the total
concentration of cells, the concentration of non-viable cells and calculation of viability
in a cell sample. Furthermore various alternatives for diluting the samples are
presented. The fundamentals on which the NucleoCounter is based are discussed
thoroughly in chapter 2 above.
The NucleoCounter method is based on staining mammalian cell DNA with a fluorescent
dye, propidium iodide. Propidium iodide is immobilized inside the NucleoCassette and
has the ability to stain DNA of non-viable cells. Propidium iodide enters non-viable cells,
as their plasma membrane is permeable. Thereby only the nuclei of non-viable cells are
stained when mixed with propidium iodide.
In order to measure the total concentration of cells the plasma membranes of all the
cells must be disrupted to render all the nuclei susceptible to staining with propidium
iodide. The disruption is achieved by treatment with a Lysis buffer
4 (Reagent A100)
followed by a Stabilizing buffer 2 (Reagent B). After treatment with the Lysis buffer and
Stabilizing buffer the cells, or the nuclei since the plasma membranes are disrupted, are
loaded into the NucleoCassette. Once inside the NucleoCassette the nuclei are stained
with propidium iodide.
Having obtained the total concentration of cells and the concentration of non-viable
cells, the viability can be calculated. This can either be done manually or automatically
by use of the NucleoView software.
The following list contains the items needed for cell counting with the NucleoCounter.
•
Cell suspension
•
Lysis buffer 4 (Reagent A100)
•
Stabilizing buffer 4 (Reagent B)
•
NucleoCassettes
•
Pipette and tips
•
Dispensettes if convenient
•
Sample vials (approx. 1,5 ml)
Remember Remember Remember Remember---- samples obtained from the cell suspension must be samples obtained from the cell suspension must be samples obtained from the cell suspension must be samples obtained from the cell suspension must be
representative. representative. representative. representative.
6.1 Total cell count
To determine the total concentration of cells draw a sample of the cell suspension into a
vial. The sample size is optional as long as at least 100µl of the stabilized lyzate is
4 Development of the Lysis buffer and Stabilizing buffer is an ongoing process. Contact
ChemoMetec A/S, or your local distributor or send an email to [email protected]
for the latest update.