DURAClone RE PC Protocol
This protocol is for reference purposes only. It may be necessary to adapt the method for specific research needs.
KIT BOX CONTENTS
25 tests of the DURAClone RE PC Antibody Panel
3 Compensation Kits, each kit containing eight single color tubes:
- CD56-APC-Alexa Fluor 750
- CD4-Pacific Blue
- CD8-Krome Orange
NOTE: Do not store the reagent tubes in the refrigerator; do not freeze/thaw the tubes. Minimize the exposure of the tubes to light, especially during processing and incubation of sample(s) prior to acquisition.
MATERIAL REQUIRED BUT NOT SUPPLIED
Blood collection tube containing anticoagulant, K2 EDTA or K3 EDTA
VersaLyse Solution (Part Number A09777)
IOTest 3 Fixative Solution (Part Number A07800).
VersaLyse Fix-and-Lyse Mixture:
- Prepared fresh each day by adding 25 μL of 10X IOTest 3 Fixative Solution to 1 mL of VersaLyse Solution. Each sample and compensation control requires 1 mL.
Phosphate Buffered Saline, PBS (Part Number 6603369, or equivalent)
- Prepared following the IFU.
- Prepared fresh each day by adding 1 mL of 1X PBS to 12.5 μL of IOTest®3 10X Concentrate. Each sample and compensation control requires 500 μL.
VersaComp Antibody Capture Beads (Part Number B22804)
Flow cytometer equipped with the following lasers and detectors:
- 405 nm: 430 – 470 nm and 530 – 570 nm
- 488 nm: 504 – 545 nm, 560 – 600 nm, 605 – 635 nm, 680 – 710 nm and >755 nm
- 633 nm: 650 – 670 nm, 715 – 735 nm and >755 nm
Flow cytometer calibration beads
- Determine the total number of leukocytes in the bone marrow samples.
- Transfer the volume of sample equivalent to 3 to 5 million leukocytes to an empty tube.
- Add 1 mL of VersaLyse Fix-and-Lyse Mixture per 100 μL of bone marrow cells. Vortex the tube at high speed for 1-3 seconds and incubate the tube for 15 minutes between 18 and 25 °C. Protect from light.
- Centrifuge the tube at 300 x g for 5 minutes; aspirate the supernatant. Gently tap to dissociate the pellet.
- Resuspend the pellet with 100 μL of PBS and transfer to the DURAClone RE PC Antibody Panel tube. Vortex at high speed for 6-8 seconds and incubate for 15 minutes between 18 and 25 °C. Protect from light.
- Add 3 mL 1X PBS; centrifuge at 150 x g for 5 minutes; aspirate the supernatant.
- Resuspend cells in 500 μL of 1X PBS/Fixative solution.
- The sample is ready for acquisition. Set the discriminator on the FS parameter to a value low enough to assure lymphocytes are not excluded from acquisition.
- Add 100 μL of whole blood to each of the single color tubes in the Compensation Kit. All tubes should be from the same pouch.
- Add one drop of the positive VersaComp Antibody Capture Beads to the following compensation tubes:
- Create a FS INT vs FS TOF dot plot and a singlet gate to eliminate any doublets (identified by higher values on FS TOF)
- Create a FSC vs. SSC dot plot and apply the singlets gate. Create a region to exclude debris (scattergate) and another region to include low SSC lymphocytes.
- Create a CD27-PE vs. CD81 FITC dot plot and apply the scattergate. Draw a region to exclude aggregates.
- Create a CD138-APC vs. SSC dot plot and apply the gate from step 3. Draw a region to gate the CD138+ SSC low cells.
- Create a CD138-APC vs CD38-Pacific Blue and apply the CD138+ gate. Create a region to encompass the CD138+CD38+ cells.
- Create a Boolean gate, which compares the low SSC lymphocytes with the CD138+CD38+ gate and apply the same on the following plots:
- Create a CD138-APC vs CD81-FITC.
- Create a CD138-APC vs CD27-PE.
- Create a CD138-APC vs CD19-PC5.5.
- Create a CD138-APC vs CD200-PC7.
- Create a CD138-APC vs CD56-APC-Alexa Fluor 750.
- Create a CD138-APC vs CD45-Krome Orange.