DURAClone IM T Cell Subsets 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 T Cell Subsets Antibody Panel

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

  • CD4-FITC
  • CD4-PE
  • CD28-ECD
  • CD279 (PD1)-PC5.5
  • CD27-PC7
  • CD4-APC
  • CD8-Alexa Fluor 700
  • CD3-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

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 T Cell Antibody Panel.

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

  3. Add 2 mL of VersaLyse, vortex the tube at high speed for 1-3 seconds and incubate 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 now ready for acquisition. Set the discriminator on the FS parameter to a value low enough to assure 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 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 a CD45-Krome Orange vs. SSC dot plot and apply the Leukocyte gate. Create a region to encompass the CD45+ lymphocytes.

  3. Create a CD3-APC-Alexa Fluor 750 vs. SSC dot plot and draw a region to gate the CD3+ T cells.

  4. Create a CD4-APC vs. CD8-Alexa Fluor 700 dot plot and draw two regions to gate the CD4+ T cells and CD8+ T cells respectively.

  5. Create four dot plots as follows, gating either on CD4+ or on CD8+ T cells may be applied to these plots:

    • Create a CD57-PacBlue (Pacific Blue) vs. SSC dot plot and draw a region to encompass the CD57+ cells.

    • Create a CD279 (PD1)- PC5.5 vs. SSC dot pot and draw a region to encompass the PD-1+ cells.

    • Create a CD45RA-FITC vs.CD197 (CCR7)-PE dot plot and draw a quadrant to delineate the following:

      • CD197 (CCR7)+ CD45RA- Central memory cells

      • CD197 (CCR7)+ CD45RA+ Naïve T cells

      • CD197 (CCR7)-CD45RA- Effector memory T cells

      • CD197 (CCR7)-CD45RA+ Effector T cells

    • Create a CD28-ECD vs. CD27-PC7 dot plot and draw a quadrant to delineate the following:

      • CD27+ CD28- cells

      • CD27+ CD28+ cells

      • CD27-CD28+ cells

      • CD27-CD28- cells

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