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Principles of Gating
Publication Name:
Current Protocols in Cytometry
Unit Number:
Unit 1.8
DOI:
10.1002/0471142956.cy0108s03
Online Posting Date:
May, 2001
Abstract
Few aspects of flow cytometry are common to every application. Gating principles is one. This unit provides an excellent overview as well as detailed conceptual material on how gating works, when certain types of gating should or should not be applied, and the advantages and disadvantages of various techniques. This unit should be required reading for every technician using a flow cytometer and every graduate student starting out in the area, and is an excellent refresher for those more advanced in the technology. It brings out the historical development of our understanding of gating applications as well as providing a high level of technical accuracy.
Figures
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Figure 1.8.1 Gating for sort purity. To achieve the expected sort purity, the threshold gate (TG) must be set low enough to trigger on all possible contaminating particles. (A) In this panel, the threshold is set higher than the amplitude of the dimmer contaminating particles. As a result, the dimmer particles are not processed by the sort-decision logic circuitry and appear as contaminants in the sorted sample. (B) In this panel, the trigger threshold is set below the dimmer particles, and they are now excluded in the sorted sample. -
Figure 1.8.2 Gating with the PRISM parameter. Lymphocytes stained with CD2, CD4, and CD8 antibodies are easily analyzed with the PRISM parameter, which shows the number of lymphocytes binding each of the eight combinations of CD2, CD4, and CD8 antibodies. -
Figure 1.8.3 Light-scatter gating is commonly used to identify lymphocytes, monocytes, and granulocytes in a whole blood preparation. -
Figure 1.8.4 Gating for doublets and multiplets. Doublets and larger multiplets are commonly detected by comparing fluorescence pulse peak amplitude to the pulse integral amplitude. In (A) a two-parameter plot of peak versus integral pulse amplitudes is used to identify the singlet events which lie along the diagonal and the multiplet events which lie below and to the right of the diagonal. An alternative approach is shown in (B) using a single-parameter histogram of the ratio of peak and integral pulse amplitudes. The singlets are identified by the peak to the right and the multiplets are found to the left of the singlet peak. -
Figure 1.8.5 Back gating can be used to check the effectiveness of a particular gate in a gating equation. In this case, back gating is used to determine the level of monocyte contamination in the lymphocyte light-scatter gate. In panel (A) the light-scatter gate is set; in panel (B) the monocyte gate (CD14 + ) is set; in panel (C) the two gates are combined. The contaminating monocytes are identified as those cells which satisfy the lymphocyte light-scatter gate and are positive for CD14. The resulting histogram shows the small number of monocytes that would contaminate the lymphocyte analysis.
Videos
Literature Cited
| Literature Cited | |
| Auer, R.E., Starling, D., Weber, B., and Wood, J.C.S. 1993. A data acquisition system for flow cytometry with wide dynamic range analog to digital conversion and digital signal processing. Cytometry (Suppl.) 6:146A. | |
| Cupp, J.E., Leary, J.F., Cernichiari, E., Wood, J.C.S., and Doherty, R.A. 1984. Rare-event analysis methods for detection of fetal red blood cells in maternal blood. Cytometry 5:138-144. | |
| Frankel, D.S., Loken, M.R., Stelzer, G.T., Shults, K.E., and Bagwell, C.B. 1993. Neural network analysis of flow cytometric data for normal and leukemic bone marrow. Cytometry (Suppl.)6:44. | |
| Leary, J.F., Ellis, S.P., and McLaughlin, S.R. 1991. 3-D autostereoscopic viewing of multidimensional data for principal component/biplot analysis and sorting. Cytometry (Suppl.)5:134-135. | |
| Loken, M.R., Brosnan, J.M., Bach, B.A., and Ault, K.A. 1990. Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry. Cytometry 11:453-459. | |
| Mandy, F.F., Bergeron, M., Recktenwald, D., and Izaguirre, C.A. 1992. A simultaneous three-color T cell subsets analysis with single laser flow cytometers using T cell gating protocol: Comparison with conventional two-color immunophenotyping method. J Immunol. Methods 156:151-156. | |
| McCoy, J.P. Jr., Chambers, W.H., Lakomy, R., Campbell, J.A., and Stewart, C.C. 1991. Sorting minor subpopulations of cells: Use of fluorescence as the triggering signal. Cytometry 12:268-274. | |
| Murphy, R.F. 1985. Automated identification of subpopulations in flow cytometric list mode data using cluster analysis. Cytometry 6:302-309. | |
| Nicholson, J.K.A., Jones, B.M., and Hubbard, M. 1993. CD4 T-lymphocyte determinations on whole blood specimens using a single-tube three-color assay. Cytometry 14:685-689. | |
| Redelman, D. 1993. Improved procedures for training neural networks to analyze flow cytometric data. Cytometry (Suppl.)6:43. | |
| Snow, C. and Bauer, K. 1994. Usefulness of DNA fluorescence ratioing in reducing DNA measurement artifacts caused by aggregates. Cytometry (Suppl.)7:50. | |
| van den Engh, G. and Stokdijk, W. 1989. Parallel processing data acquisition system for multilaser flow cytometry and cell sorting. Cytometry 10:289-293. | |
| Key Reference | |
| Shapiro, H.M. 1995. Practical Flow Cytometry, 3rd ed. Wiley-Liss, New York. | |
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A resource for more examples of the use of gating in flow cytometry applications. | |
