B60231–AE
5 of 128
Reagents & Samples
Volume
1X NH
4
Cl Lysing Solution
2 mL
Vortex – Incubate at room temperature for 10 minutes. Protect from light
Stem-Count Fluorospheres
100 µL
MANUAL GATING AND ANALYSIS METHOD
Protocol Setup
The flow cytometer must be equipped to detect Forward Scatter, Side Scatter and four fluorescence channels. For
the FL3 channel (for Stem-Count or Flow-Count Fluorospheres monitoring) use a 620 nm band pass filter. For the
FL4 channel (for 7-AAD Viability Dye monitoring) use a 675 nm long pass filter.
NOTE:
The same gating scheme and series of 8 histograms as stated for specimen analysis,
must be followed for Stem-Trol Control Cells. As Stem-Trol Control Cells have size
characteristics close to, and express CD45 and CD34 antigens at densities that
approximate, normal immature hematopoietic cells, there is no need to modify the region
boundaries along the Forward Scatter, Fluorescence 1 and Fluorescence 2 channels.
However as Stem-Trol Control Cells Side Scatter characteristics are unique you may
adjust the region boundaries along the Side Scatter. Regions A, B, C, and D (see the
heading Region Creation for region definition) must be modified to include the Stem-Trol
Control Cells characteristic cluster.
Histogram Creation
Create histograms as follows:
1. Create Histogram 1 as FL1 CD45-FITC vs Side Scatter.
2. Create Histogram 2 as FL2 CD34-PE vs Side Scatter.
3. Create Histogram 3 as FL1 CD45-FITC vs Side Scatter.
4. Create Histogram 4 as Forward Scatter vs Side Scatter.
5. Create Histogram 5 as FL1 CD45-FITC vs FL2 CD34-PE.
6. Create Histogram 6 as Forward Scatter vs Side Scatter.
7. Create Histogram 7 as Time vs FL3 Stem-Count or Flow-Count Fluorospheres.
8. Create Histogram 8 as FL4 7-AAD vs Side Scatter.
Histograms 1 to 4 are intended to characterize CD34
+
HPC, a process that may be delayed until the analysis step.
These first four histograms are set up according to the ISHAGE Guidelines for CD34
+
Cell Determination by Flow
Histograms 5 to 7 are intended to monitor parameters that are of importance during the acquisition step. These
include the Forward Scatter discriminator, the number of CD45
+
events to be collected and the correct fluorosphere
singlets accumulation.
Histogram 8 is intended to discriminate and analyze viable events from nonviable events when required.
Region Creation
Create regions as follows:
1. Histogram 1 – Create a rectilinear Region A to include all CD45
+
leukocytes and eliminate platelets, red blood
cell debris, and aggregates.
2. Histogram 1 – Create an amorphous Region E on the lymphocytes (bright CD45, low side scatter).
3. Histogram 2 – Create a rectilinear Region B on Histogram 2 to include all CD34
+
events with low to intermediate
Side Scatter. Set a stop count of 75,000 events (CD45
+
events) in Histogram 2.
4. Histogram 3 – Create an amorphous Region C on Histogram 3 to include all clustered CD45
dim
events.
5. Histogram 4 – Create an amorphous Region D on Histogram 4 to include all clustered events with intermediate
side scatter and intermediate to high forward scatter.
6. Histogram 5 – Create a Quadstat Region I on Histogram 5 to verify the lower limit of CD45 expression on
CD34
+
events.
