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NOTE:
In order to standardize the analysis, when receiving new Stem-Kit Reagents, and at
daily intervals afterwards, run a process check by staining Stem-Trol Control Cells with
a normal peripheral blood sample from a healthy donor. Furthermore, as Stem-Trol
Control Cells are stabilized cells (i.e. nonviable cells), 7-AAD Viability Dye staining may
be visually checked on Stem-Trol Control Cells (see the heading: Histogram Creation).
Ensure that the flow cytometer is properly aligned and standardized for fluorescence intensity according to the
manufacturer and laboratory guidelines. Ensure that fluorescence compensation settings are properly adjusted
according to the manufacturer and laboratory guidelines.
Bring the control reagent and antibodies to room temperature.
For each experiment, label one tube: TROL 45/34/7-AAD.
1. Pipet 20 µL of CD45-FITC / CD34-PE into the tube.
2. Pipet 20 µL of 7-AAD Viability Dye into the tube.
IMPORTANT:
Risk of incomplete lysis if blood specimen remains on the top or side of the test tube.
Use care when pipetting to prevent the blood from touching the top or side of the test
tube. Clean the tube with a cotton swab, if necessary, to remove all traces of blood
specimen from the top or side of the test tube.
3. Into the bottom of the tube, accurately pipet 100 µL of a well-mixed normal whole blood sample using a
calibrated positive displacement or repeater pipet.
4. Prepare and add Stem-Trol:
•
Vortex Stem-Trol Control Cells for 5 seconds.
•
Accurately pipet 20 µL of Stem-Trol Control Cells into the tube.
•
Vortex tubes for 5 seconds.
5. Incubate at room temperature (18 – 25°C) for 20 minutes, protected from light.
6. Add 2 mL of prepared 1X NH
4
Cl Lysing Solution into tube and vortex immediately for 5 seconds.
For details on preparing the Lysing Solution, refer to the Stem-Kit Reagents package insert.
7. Incubate at room temperature for 10 minutes, protected from light.
8. Store the tubes in a rack placed on ice (2 – 8°C), and protected from light.
IMPORTANT:
Risk of erroneous results if air bubbles are pipetted. Excessive mixing of Stem-Count
or Flow-Count Fluorospheres can create air bubbles. Do not excessively mix the
Stem-Count or Flow-Count Fluorospheres, and do not pipette air bubbles into the
sample tubes.
9. Gently mix the Stem-Count or Flow-Count Fluorospheres by inverting the vial 3 to 5 times before using. Avoid
excessive mixing to minimize air bubble formation.
10.Prior to acquisition, pipette 100 µL of Stem-Count or Flow-Count Fluorospheres into the tube.
11. Vortex for 5 seconds immediately after each addition. Store at 2 – 8°C. Repeat vortexing immediately prior to
flow cytometric acquisition.
IMPORTANT:
Risk of erroneous results if sample is analyzed more than 1 hour after adding
Stem-Count or Flow-Count Fluorospheres. Prepared samples must be analyzed within 1
hour of adding Stem-Count or Flow-Count Fluorospheres.
Preparation Summary (Tube Label: TROL45/34/7-AAD)
Reagents & Samples
Volume
CD45-FITC / CD34-PE
20 µL
7-AAD Viability Dye
20 µL
Whole Blood
100 µL
Stem-Trol Control Cells
20 µL
Vortex – Incubate at room temperature for 20 minutes. Protect from light
