DURAClone IM Phenotyping Basic 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 Phenotyping Basic Antibody Panel

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

  • CD4-FITC
  • CD4-PE
  • CD19-ECD
  • CD14-PC7
  • CD4-APC
  • CD8-Alexa Fluor 700
  • CD3-APC-Alexa Fluor 750
  • 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

Calibrated pipettes

Vortex mixer

VersaLyse Solution (Part Number A09777)

IOTest 3 Fixative Solution (Part Number A07800)

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.

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 100 μL of whole blood to one tube of the DURAClone IM Phenotyping Basic Antibody Panel.

  2. Vortex at high speed for 6-8 seconds and incubate for 15 minutes between 20 and 30 °C. Protect from light.

  3. Add 2 mL of VersaLyse, vortex at high speed for 1-3 seconds and incubate the tube for 15 minutes between 20 and 30 °C. Protect from light.

  4. Centrifuge the tube at 200 x g for 5 minutes; aspirate the supernatant. Gently tap to dissociate the pellet.

  5. Add 3 mL 1X PBS; centrifuge at 200 x g for 5 minutes. Aspirate the supernatant. Gently tap to dissociate the pellet.

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

  7. The sample is ready for acquisition. Set the discriminator on the FS parameter such that the lymphocytes are not excluded from the 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. Follow steps steps 2-7 in the Sample Preparation procedure.  

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

Analysis

  1. Create a CD45-Krome Orange vs. SSC dot plot and create a region to encompass the CD45+ leukocytes.

  2. Create three plots as follows:

    • Create a CD14- PC7 vs. SSC dot plot and apply the CD45+leukocyte gate onto this plot and draw a region to encompass the CD14+ cells. These cells are the monocytes.

    • Create a CD19- ECD vs. CD3-APC-Alexa Fluor 750 dot pot and draw a region to encompass the CD19+ cells. These cells are the B lymphocytes.

    • Create a CD56-PE vs CD3-APC-Alexa Fluor 750 dot plot. Create a Boolean gate "Lymphocytes AND (NOT CD19+)" and apply this gate. Draw a region to encompass the CD56+ and CD3+ cell populations. Draw as region to encompass the CD56+ and CD3+ cell populations. The CD56+ cells are the Natural Killer cells and the CD3+ cells are the T lymphocytes (T cells).

  3. Create a CD56-PE vs. CD16-FITC dot plot.

    • Apply the CD56+ gate from the CD56-PE vs. CD3-APC-Alexa Fluor 750 dot plot.

    • Draw a region to encompass the CD56+ high NK cells.

  4. Create a CD4-APC vs. CD8-Alexa Fluor 700 dot plot.

    • Apply the gate CD3+ T cells gate from the CD56- PE vs.CD3- APC-Alexa Fluor 750 dot plot.

    • Draw a region to encompass the CD4+ T cells and another region to encompass the CD8+ T cells.

  5. Create CD16-FITC vs. CD14-PC7 dot plot.

    • Apply the CD14+ gate onto this plot.

    • Add regions to encompass the following cell populations:

      • CD 14+ high CD16- monocytes

      • CD14 + high CD16+ monocytes

      • CD14+ CD16 high monocytes

  6. Record the desired statistics of all the gated cell populations.