B60231–AE
6 of 128
7. Histogram 5 – Create an amorphous Region H on Histogram 5 to surround all Stem-Count or Flow-Count
Fluorospheres, including doublets. The region H should be located at the top right corner of the Histogram 5.
NOTE:
Be sure that Region H is drawn as an AMORPHOUS region.
8. Histogram 6 – Copy Region D from Histogram 4 as Region F in Histogram 6.
9. Histogram 7 – Create a rectilinear Region G on Histogram 7 to include the Stem-Count or Flow-Count
Fluorospheres singlets only. Region G can be labelled as “ CAL ” to allow automatic cal culation of absolute
numbers of CD34
+
HPC (refer to the instrument manual for further details).
10.Histogram 8 – Create a rectilinear Region J on Histogram 8 to separate viable leukocytes (7- AAD negative
events) from non viable events (mainly Stem-Trol Control Cells positive for 7-AAD staining).
Gate Creation
Create gates as follows:
1. Histogram 1 – Assign “H” to Histogram 1 to display all events excluding all Stem-Count or Flow-Count
Fluorospheres. Refer to the instrument manual for the creation of “ not gates. ”
2. Histogram 2 – Assign “A” to Histogram 2 to display all CD45
+
events.
3. Histogram 3 – Assign “A” and “ B ” (AB) to Histogram 3 to display all CD45
+
CD34
+
events.
4. Histogram 4 – Assign “A” and “B” and “C” (ABC) to Histogram 4 to display all CD45
+
CD34
+
events clustered
events with low to intermediate side scatter and low CD45 staining expression. Events from Region ABCD
are real CD34
+
HPC.
5. Histogram 5 – Ungated to display all events.
6. Histogram 6 – Assign “E” to display lymphocytes as a visual check on the discriminator.
7. Histogram 7 – Assign “H” to histogram 7 to display all Stem-Count or Flow-Count Fluorospheres, including
doublets.
8. Histogram 8 – Assign “A” to histogram 8 to display CD45
+
events.
Flow Cytometer Setting
1. Ensure that the flow cytometer is properly aligned and standardized for light scatter and fluorescence intensity
according to the manufacturer’s and laboratory guidelines. Verify that color compensation is set for standard
operation. Refer to the instrument’s manual for further instructions.
2. Vortex test tubes for 5 seconds.
3. Perform data acquisition on the flow cytometer. A minimum of 75,000 CD45
+
events must be analyzed.
4. Adjust the discriminator and regions by analyzing the TROL 45/34/7-AAD tube.
Analysis Example
The histograms shown in the APPENDIX are displayed in an ascending number order as displayed on the protocol.
Calculation of CD34
+
Stem-Trol Control Cells
Using flow cytometric results automatically adjusted with the Stem-Count or Flow-Count Fluorospheres Assayed
Concentration obtained using System II Software (Version 3.0) and a COULTER EPICS XL/XLMCL flow cytometer.
In order to obtain automatically calculated absolute count determinations on COULTER EPICS XL / XL-MCL flow
cytometers, the correct Stem-Count or Flow-Count Fluorospheres Assayed Concentration must be entered before
sample acquisition.
Enter "CAL" as the Name of the Stem-Count or Flow-Count Fluorospheres singlets Region G, and enter the
value, for example, 1,000 into the CAL FACTOR box on the STATISTICS dialog screen of the SET-UP SCREEN
PROTOCOL menu.
Entering the CAL Factor for Stem-Count or Flow-Count Fluorospheres:
1. At the Acquisition Run screen, select Setup Screen >> Protocol.
2. Select Statistics >> CAL FACTOR.
3. Enter the CAL factor number (assayed concentration) from the Stem-Count or Flow-Count Fluorospheres vial.
4. Press ENTER.
5. Select OKAY.
6. At the prompt, type Y for yes.
