BD LSR II Kullanıcı Kılavuzu - Sayfa 5

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3. Diva will create a new specimen called "Compensation Controls". Expand the specimen and activate

the first tube.

4. Set Events to display to 5000.

5. Run unstained cells or beads first.

6. Adjust the FSC and SSC voltages to place the population of interest on scale.

7. Adjust the FSC threshold value to exclude most of the debris without excluding the population of

interest.

Setting it to 50% of the smallest cell of interest works well.

8. Move the P1 gate on the FSC/SSC plot to include your cells of interest.

9. Put your fully stained sample on. Make sure the positives are on scale. If your positives are above 10

you will need to decrease the voltage.

*Remember, if you significantly lower the PMT voltage below the baseline settings in order to bring the

positive population on scale, then the dim populations might not be easily resolved from the negative

populations for that parameter. It is acceptable to have the negative peak shift above the first log decade in

10. Check each single color in the unstained cell tube area so we can define that all events are on scale.

11. Run and record each of your single-stained controls. Use the P2 gate to select only your positive peak.

Use an interval gate to create a P3 gate around your negative peak.

12. Select Experiment à Compensation Setup à Calculate Compensation. When the Single Stained Setup

box appears, select "Apply Only" to apply the calculated values to your experiment.

some detectors.