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

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 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. Draw a region around the CD45+/low SSC Lymphs cell population. Draw a region around the CD45+/high SSC Granulocytes cell population.
  4. Create three dot plots as follows:
    • Create a CD14- PC7 vs. SSC dot plot and apply the Leukocytes gate onto this plot. Draw a region to encompass the CD14+ cells. These cells are the CD14+ Monocytes.
    • Create a CD56-PE vs CD3-APC-Alexa Fluor 750 dot plot and apply the Lymphs gate onto this plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CD56+ and CD3+ cell populations. The CD56+/CD3- cells are the Natural Killer (NK) cells, the CD56+/CD3+ cells are the NKT like cells and the CD3+/CD56- cells are the T lymphocytes (T cells).
    • Create a CD19- ECD vs. CD3-APC-Alexa Fluor 750 dot plot and apply the Lymphs gate onto this plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CD3+ and CD19+ cell populations. These cells are the CD3+ T lymphocytes and CD19+ B lymphocytes.
  5. Create a CD4-APC vs. CD8-Alexa Fluor 700 dot plot.
    • Apply the CD3+ T cells gate from the CD19- ECD vs.CD3- APC-Alexa Fluor 750 dot plot.
    • Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CD4+ T cells, CD8+ T cells, double positive (DP) T cells and double negative (DN) T cells.
  6. Create CD16-FITC vs. CD14-PC7 dot plot.
    • Apply the CD14+ Monocytes gate onto this plot.
    • Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the following cell populations:
      • CD14+ high CD16- population. These cells are the Classical monocytes.
      • CD14+ high CD16+ population. These cells are the Intermediate monocytes.
      • CD14+ low CD16+ high population. These cells are the non-classical monocytes.
  7. Create a CD56-PE vs. CD16-FITC dot plot.
    • Apply the CD56+ NK cells gate from the CD56-PE vs. CD3-APC-Alexa Fluor 750 dot plot.
    • Draw a region to encompass the CD56+ high NK cells.
  8. Record the desired statistics.