DURAClone IM Granulocytes 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 IM Granulocytes Antibody Panel

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

  • CD4-FITC
  • CD16-ECD
  • CD33-PC5.5
  • CD11b-PC7
  • CD4-APC
  • CD19-APC-Alexa Fluor 700
  • CD62L-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

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 and compensation control requires 2 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 – 570nm
  • 488 nm: 504 – 545 nm, 605 – 635 nm, 680 – 710 nm and >755nm
  • 633 nm: 650 – 670 nm, 715 – 735 nm and >755 nm

Sheath fluid

Flow cytometer calibration beads

Staining Procedure

SAMPLE PREPARATION

  1. Add 100 μL of whole blood to one tube of the DURAClone IM Granulocytes 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 2 mL of VersaLyse Fix-and-Lyse Mixture. Vortex at high for 1-3 seconds and incubate for 15 minutes between 18 and 25 °C. Protect from light.
  4. Centrifuge the tube at 200 x g for 5 minutes; aspirate the supernatant. Vortex at high speed for 1-3 seconds to dissociate pellet.
  5. Add 3 mL 1X PBS; centrifuge at 200 x g for 5 minutes. Aspirate the supernatant. Vortex at high speed for 1-3 seconds to dissociate pellet.
  6. Resuspend cells in 500 μL of 1X PBS/Fixative solution.
  7. Sample is ready for 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 a single pouch.
  2. Add one drop of the positive VersaComp Antibody Capture Beads to the following compensation tubes:
    • CD16-ECD
    • CD33-PC5.5
    • CD11b-PC7
    • CD19-APC-Alexa Fluor 700
    • CD62L-APC-Alexa Fluor 750
  3. Follow steps 2-7 in the Sample Preparation procedure.  
  4. Follow standard procedures and instrument manufacturer instructions for compensation setup.

Analysis

Example Data in Kaluza Analysis File (download)

  1. Create an FSC-A vs. FSC-H dot plot and draw a region to encompass the singlets cell population and exclude the doublets. 
  2. Create a FSC-A vs. SSC-A dot plot and apply the singlets gate onto this plot. Draw a region to encompass the Cells population. These are the singlets cell populations. 
  3. Create a CD45-Krome Orange vs. SSC-A dot plot and apply the Cells gate onto this plot. Draw a region to encompass the CD45+ leukocytes. These are the singlet CD45+ Leukocytes cells.
  4. Create a 3-19-56-14-Lineage-APC-Alexa Fluor 700 vs. CD294-FITC dot plot and apply the Leukocytes gate onto the plot. Create a region to encompass the Lineage- CD294+ cells and name the region “CD294+”. Create another region to encompass the Lineage+ CD294- gate and name the region “CD294-“.
  5. Create a CD15-Pacific Blue (PB) vs SSC-A dot plot and apply the CD294+ gate from the 3-19-56-14-Lineage-APC-Alexa Fluor 700 vs. CD294-FITC dot plot. Create a region to encompass the CD15+ SSC high cells. These are the Eosinophils. Create another region on the plot to encompass the CD15- SSC low cells. These are the Basophils.
  6. Create a 3-19-56-14-Lineage-APC-Alexa Fluor 700 vs. CD15-Pacific Blue (PB) dot plot and apply the CD294- gate from the 3-19-56-14-Lineage-APC-Alexa Fluor 700 vs. CD294-FITC dot plot. Create a region to encompass the Lineage- CD15+ cells. These are the CD15+ Neutrophils.
  7. Create a CD16-ECD vs. CD62L-APC-Alexa Fluor 750 dot plot and apply the CD15+ Neutrophils gate onto the dot plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CD16+ CD62L low neutrophils, CD16+ CD62L+ high mature neutrophils and CD16- CD62L+ high neutrophils. 
  8. Create a CD11b-PC7 vs. CD274-APC dot plot and apply the CD15+ Neutrophils gate onto the dot plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CD11b low CD274- neutrophils and the CD11b+ high CD274+ neutrophils. 
  9. Create a 3-19-56-14-Lineage-APC-Alexa Fluor 700 vs. CD33-PC5.5 dot plot. Create a “No Neutrophils” Boolean gate by combining gates using the formula: Leukocytes AND (NOT “CD15+ Neutrophils”)) AND CD294-. Apply the No Neutrophils Boolean gate to this plot. Draw a region to encompass the Lineage- CD33+ population. These are the CD33+ Monocytes. 
  10. Create a CD16-ECD vs. 3-19-56-14-Lineage-APC-Alexa Fluor 700 dot plot and apply the CD33+ gate onto this plot.  Create regions to encompass the CD16- CD14+ classical monocytes, CD16+ CD14+ intermediate monocytes and CD16+ CD14 dim+ non-classical monocytes.
  11. Create a CD11b-PC7 vs. CD274-APC dot plot and apply the CD294+ gate from the 3-19-56-14-Lineage-APC-Alexa Fluor 700 vs. CD294-FITC onto the plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to encompass the CD11b+ high CD274- population. These quadrant line locations will be used as a control to delineate the CD11b+ low CD274- and CD11b+ high CD274+ monocytes in a subsequent dot plot. 
  12. Create another CD11b-PC7 vs. CD274-APC dot plot. Create a “Monocytes” Boolean gate by combining all CD33+ monocyte regions using the formula: (classical or intermediate) or non-classical. Apply the Monocytes Boolean gate to this plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CD11b+ low CD274- MO (monocytes) and the CD11b+ CD274+ MO (monocytes).  
  13. Record the desired statistics.