Excluding Nonspecific Labeled Cells by Using a Dump Channel

To optimize the analysis, the best possible labeling strategy should be selected. A one-antibody strategy for identification of a certain population may be improved upon by using a second antibody that positively identifies the cells and by using a so-called dump channel. An example of the latter is shown in Figure 1. In Figure 1a, whole blood was labeled with an anti-blood dendritic cell antigen-2 (BDCA-2 or CD303) monoclonal. This antigen is exclusively expressed by plasmacytoid DCs in human blood, but it is not entirely clear where a gate defining positive cells should be set. In the next plot (Fig. 1B), a dump channel was used: a cocktail of antibodies all conjugated to the same fluorochrome and all specific for antigens that are not expressed by plasmacy-toid cells. An anti-HLA-DR monoclonal was also included, and it is quite easy to distinguish a DR-positive, phycoerythrin (PE)-negative population. This population could then be gated for BDCA-2-positive and -negative events or,

Fig. 1. Using a dump channel to identify plasmacytoid dendritic cells (DCs) in peripheral blood. (A-D) Whole blood was labeled to identify plasmacytoid DCs. A single antibody specific for these cells BDCA2+ is compared with the use of a dump channel together with anti-CD123, which is expressed on plasmacytoid DLs. After labeling, erythrocytes were lysed and white cells were fixed before analysis on a FACScan. (A) This dot plot shows BDCA-2-labeled cells; the side light scatter versus BDCA-2-PE profile is shown. A gate was set around the cluster of BDCA-2-positive cells, but background and/or possibly positive cells made it difficult to define truly positive cells. The histogram in (C) shows that this antibody does give a good result; these cells were labeled also with HLA-DR-PerCP (peridinin chlorophyll protein) and were gated on DR+ BDCA+ cells. (B) and (D) show whole blood labeled with CD123-FITC,

Fig. 1. Using a dump channel to identify plasmacytoid dendritic cells (DCs) in peripheral blood. (A-D) Whole blood was labeled to identify plasmacytoid DCs. A single antibody specific for these cells BDCA2+ is compared with the use of a dump channel together with anti-CD123, which is expressed on plasmacytoid DLs. After labeling, erythrocytes were lysed and white cells were fixed before analysis on a FACScan. (A) This dot plot shows BDCA-2-labeled cells; the side light scatter versus BDCA-2-PE profile is shown. A gate was set around the cluster of BDCA-2-positive cells, but background and/or possibly positive cells made it difficult to define truly positive cells. The histogram in (C) shows that this antibody does give a good result; these cells were labeled also with HLA-DR-PerCP (peridinin chlorophyll protein) and were gated on DR+ BDCA+ cells. (B) and (D) show whole blood labeled with CD123-FITC, as shown in Figure 1C, CD123-positive events, which thus defined the plasma-cytoid DC population.

Unspecific labeling is most common for Fc receptor-positive cells, dead and dying cells, and debris. For this reason, CD14, CD16, and CD56 may be useful antigens to target unless, of course, they are expressed by the cells of interest. It may also be desirable to use a dye to exclude dead cells or erythrocytes.

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Responses

  • isaias
    What does dump channel do in flow?
    3 years ago
  • sherry cox
    How to make dump gate in flow cytometry?
    2 years ago
  • JESSAMINE
    How does dump strategy exclude certain cells?
    5 months ago

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