Analysis of PI stained tachyzoites using flow cytometry. Protocol: JRadke060601 Ó2001 Michael W. White-Department of Veterinary Molecular Biology || questions@montana.edu
WhiteProtocols2001
1.0 Analyses of PI stained tachyzoites using flow cytometry. 1.1 Nuclear DNA content can be measured by flow cytometry using PI fluorescence. Collect fluorescence in linear mode (FL2-H vs FSC, 10000 events) and calibrate the detector for G1 (1N) parasites using an asynchronous tachyzoite population. The tachyzoite genome is small and thus the difference in fluorescence between a 1N and 2N population is also small. For this reason, the detector must be checked regularly against an asynchronous population ‘control’ fixed and stained as above. All samples should be analyzed at the same flow rate. Tachyzoites are smaller than the excitation beam, and thus higher flow rates will increase fluorescence that can lead to inconsistencies from sample to sample. We have found that a flow rate of » 300/sec is optimal.
Ó2001 Michael W. White-Department of Veterinary Molecular Biology || questions@montana.edu