Cds Fitc

Figure 6.8.9 Universal Template on a FACSCalibur. Analysis with a 4-color panel in one tube. The upper panel describes the absolute count with bright CD45 gating (Region A). The lower panel illustrates the percentage lymphocyte number with T-gate (Region B).

Dot plot no. 5: CD3 versus CD4, ungated; histogram no. 5: CD3, ungated

4. To define a fluorosphere region to permit the calculation of absolute cell numbers, create a fluorosphere region (R4;E) on dot plot no. 5 or set cursor (M1;E) on histogram no. 5

The fluorescence signal emitted by the counting microspheres will appear in several fluorescence detectors (PMTs). Flow-Count and TruCount microspheres emit different levels of fluoresence intensity. The operator must select the most appropriate color PMT or combination of PMTs that provides the best fluorescence resolution between microspheres and cells of interest.

Dot plot no. 6: CD45 versus side scatter, gated on (R1*R2;AB)

5. To assist the operator with the analysis of difficult specimens which may present poor resolution between lymphocytes and monocytes, create a region (R3;G) around the CD3 clusters. (R3;G) should include at least 97% of the T cells.

The single cluster that appears on dot plot no. 6 is used to draw the contour of the gate.

Dot plot no. 6 represents only T cells, as it is gated on CD45 and CD3. Care must be exercised when drawing the gate region (R3; G) around the cluster.

6. To report CD3+CD4+ lymphocyte percentages:

a. Set quadrants;

b. Collect from quadrant (upper right; H2) the CD3+CD4+ lymphocyte percentages.

Monocyte contamination can be monitored from the quadrant (upper left; H1).

Contamination should not exceed 3%. If it exceeds 3%, the region (R3;G) of dot plot no.6 must be redrawn.

7. To report CD3+CD8+ lymphocyte percentages:

a. Set quadrants b. Collect from quadrant (upper right;I2) the CD3+CD8+ lymphocyte percentages

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