Chapter 4: DNA Analysis 77
Doublet discrimination, or the ability to distinguish singlets from aggregates, is
also important for DNA experiments. Since doublets of G
0
/G
1
cells have the
same amount of DNA fluorescence as singlet G
2
+M cells, they accumulate in the
same fluorescence area channels (Figure 4-1 on page 76). Therefore, singlets and
doublets must be distinguished to obtain cell-cycle analysis accuracy. Signal
width vs area can be employed to accurately identify aggregate events.
DNA Setup
In this chapter, you use the BD™ DNA QC Particles kit to verify critical DNA
analysis criteria, and optimize your cytometer for DNA experiments. The
instructions given here assume that DAPI is being used as described in How to
Use DAPI with DNA QC, below. If you are using PI for DNA QC, see How to
Use PI with DNA QC, and substitute PI for DAPI in subsequent instructions.
How to Use DAPI with DNA QC
Before beginning this chapter, do the following.
• Prepare biological standards for instrument quality control using the
BD DNA QC Particles kit. Substitute the PI solution in the BD DNA QC
Particles kit with a 1.0-nanomolar (nM) DAPI solution prepared in
1% BSA.
• Prepare one tube each of chicken erythrocyte nuclei (CEN) and calf
thymocyte nuclei (CTN) sample according to the kit instructions. Substitute
the DAPI solution for the PI solution.
- The CEN sample is used to check instrument resolution (CV) and
linearity.
- The CTN sample is used to verify the system’s ability to resolve singlets
from aggregates.
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