DURAClone RE CLB Protocol

This protocol is for reference purposes only.  It may be necessary to adapt the method for specific research needs.

Materials

KIT BOX CONTENTS

25 tests of the DURAClone RE CLB Antibody Panel

3 Compensation Kits, each kit containing eight single color tubes:

  • CD4-FITC
  • CD4-PE
  • CD79b-PC5.5
  • CD19-PC7
  • CD4-APC
  • CD43-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

Calibrated pipettes

Vortex mixer

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 requires 3 mL and each compensation control requires 1 mL.

Phosphate Buffered Saline, PBS (Part Number 6603369, or equivalent)

  •  Prepared following the IFU.

1X PBS/Fixative:

  • 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:

  • FSC/SSC
  • 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

Sheath fluid

Flow cytometer calibration beads

Staining Procedure

SAMPLE PREPARATION 

  1. Add 300 μL of whole blood to one tube of the DURAClone RE CLB Antibody Panel.

  2. Vortex at high speed for 6-8  seconds and incubate for 15  minutes between 18 and 25 °C. Protect from light.

  3. Add 3 mL of VersaLyse Fix-and-Lyse Mixture. Vortex at high speed for 1-3 seconds and incubate for 15 minutes between 18 and 25 °C. Protect from light.

  4. Centrifuge the tube at 150 x g for 5 minutes; aspirate the supernatant. Gently tap to dissociate the pellet.

  5. Add 3 mL 1X PBS; centrifuge at 150 x g for 5  minutes. Aspirate the supernatant.

  6. Resuspend cells in 500  μL of 1X  PBS/Fixative solution.

  7. The sample is now ready for acquisition. Set the discriminator on the FS parameter to a value low enough to assure lymphocytes are not excluded from acquisition.

COMPENSATION SETUP

  1. 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.

  2. Add one drop of the positive VersaComp Antibody Capture Beads to the following compensation tubes:

    • CD79b-PC5.5

  3. Follow steps 2-7 in the Sample Preparation procedure.

  4. Follow standard procedures and instrument manufacturer instructions for compensation setup.

Analysis


  1. Create a FS INT vs FS TOF dot plot and a singlet gate to eliminate any doublets (identified by higher values on FS TOF).

  2. Create a CD45-Krome Orange vs. SSC dot plot and apply the singlets gate. Create a region to encompass the CD45+ lymphocytes.

  3. Create a CD19-PC7 vs. CD81-FITC dot plot and apply the low SSC gate. Draw a region to exclude aggregates (without autofluorescence/aggregates).

  4. Create a CD19-PC7 vs. SSC dot plot and apply gate from step 5 (without autofluorescence/aggregates). Draw a region to gate the CD19+ cells.

  5. Create a CD19-PC7 vs ROR1-PE and apply the CD19+ gate. Create a region to encompass the CD19+ROR1+ cells.

  6. Create a CD5-APC vs CD43-APC-Alexa Fluor 750 and apply the CD19+ROR1+ gate. Create a region to encompass the CD5+CD43+ cells.

  7. Create a CD20-Pacific Blue vs CD81-FITC and apply the CD5+CD43+ gate. Create a region to encompass the CD20- CD81- cells.

  8. Create a CD20-Pacific Blue vs CD79b-PC5.5 and apply the CD20- CD81- gate. Create a region to encompass the CD20- CD79b- cells.