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

  1. Create a FS INT vs FS PEAK dot plot and a singlet gate to eliminate any doublets (identified by values not coincident with the diagonal populations).

  2. Create a FSC vs. SSC dot plot and apply the singlets gate. Create a region to exclude debris (scatter gate)

  3. Create a Lineage (CD3-, CD19-, CD14-, CD56-APC-Alexa Fluor 700) vs. CD294-FITC dot plot and apply the scatter gate. Create a region to encompass the CD294+ Lineage- cells. Create a Boolean gate NOT(CD294+Lin-).

  4. Create a CD15-Pacific Blue vs SSC dot plot and apply the CD294+ gate. Create CD15+SSChigh gate (Eosinophils) and a CD15-SSClow gate (Basophils).

  5. Create a Lineage vs. CD15-PBE dot plot and apply the NOT(CD294+Lineage-) gate. Create a region to encompass the CD15+ Lineage- cells: CD15+. Create a Boolean gate NOT(CD15+Lineage-).

  6. Create a Boolean gate: All granulocytes = CD15+ OR (Eosinophils OR Basophils). Create a CD62L-APC-Alexa Fluor 750 vs CD16-ECD dot plot and a CD274-APC vs CD11b- PC7 dot plot and apply the All granulocytes gate.

  7. Create a Lin vs CD33-PC5.5 dot plot and apply a Boolean gate (NOT CD294+) AND (NOT CD15+) and create a region to encompass the cells. Name the region CD33+.

  8. Create a CD45-Krome Orange vs CD33-PC5.5 dot pot and apply the CD33+ gate. Create a region to encompass the cells so as to exclude the outliers from the central population. Name the region Mono.

  9. Create a CD16-ECD vs Lineage dot plot and apply the Mono gate. Create regions to encompass the classical monocytes, non-classical monocytes, CD14dimCD16- monocytes and doublets of monocytes and granulocytes.