DURAClone IM TCRs 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 TCRs Antibody Panel

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

  • CD4-FITC
  • CD4-PE
  • HLA-DR-ECD
  • VD1-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, KEDTA

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 900 μL of 1X PBS to 100 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 – 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 TCRs 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 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 the tube at 200 x g for 5  minutes. Aspirate the supernatant.

  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 to a value low enough to assure lymphocytes are not excluded from 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. Add two drops of the positive VersaComp Antibody Capture Beads to the following compensation tube:

    • VD1-PC7

  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 CD45-Krome Orange vs. SSC dot plot and apply the leukocyte gate. Create a region to encompass the CD45+ lymphocytes.

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

  3. Create the following dot plots and apply the CD3+ gate on these plots:

    • Create a HLA-DR-ECD vs. SSC plot and gate the HLA-DR+ population.

    • 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.

    • Create a TCR γδ –FITC vs. TCR αβ –PE plot and gate the TCR γδ + T cells and TCR αβ + T cells.

  4. Create a VD1-PC7 (i.e. TCR Vδ1) vs. VD2-PB (i.e. TCR Vδ2) plot, apply the TCR γδ + T cells gate on this plot and delineate the TCR Vδ1 + T cells and TCR Vδ2+ T cells.

  5. Create a CD4-APC vs. CD8-Alexa Fluor 700 plot, apply the TCR αβ + T cells gate onto this plot, and delineate the TCR αβ + CD8+ T cells and TCR αβ + CD4+ T cells.