DURAClone IM Treg 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 Treg Antibody Panel:

  • Tube 1
  • Tube 2

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

  • CD4-FITC
  • CD4-PE
  • CD39-PC5.5
  • CD4-PC7
  • FoxP3-Alexa Fluor 647
  • 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, K2 EDTA

Calibrated pipettes

Vortex mixer

PerFix-nc Cellular Staining Preparation Kit (Part Number B10825):

  • Buffer 1, fixative reagent
  • Buffer 2, permeabilizing reagent
  • Buffer 3, final solution, 10X concentrate.  Dilute to 1X prior to use.

Phosphate Buffered Saline, PBS (Part Number 6603369, or equivalent)

  •  Prepared following the IFU.

Fetal Bovine Serum

VersaComp Antibody Capture Bead Kit (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 50 μL of whole blood to one tube of the DURAClone IM Treg Tube 1.
  2. Vortex at high speed for 6-8 seconds and incubate for 15 minutes between 20 to 30 °C. Protect from light.
  3. Add 3 mL of 1X PBS; centrifuge at  500 x g for 5 minutes. Aspirate the supernatant. Gently vortex to dissociate the pellet.
  4. Resuspend cells in 50 μL 100% fetal calf serum.
  5. Add 5 μL PerFix-nc Buffer 1 and vortex at high speed for 6-8 seconds.
  6. Incubate Tube 1 for 15 minutes between 20 to 30 °C. Protect from light.
  7. Add 400 μL of PerFix nc Buffer 2 and vortex at high speed for 6-8 seconds.
  8. Transfer the contents of Tube 1 to DURAClone IM Treg Tube 2.
  9. Vortex at high speed for 6-8 seconds. Incubate for 60 minutes between 20 to 30 °C. Protect from light.
  10. Add 3 mL 1X PBS and incubate for 5 minutes between 20 to 30 °C. Protect from light.
  11. Centrifuge the tube at 500 x g for 5 minutes at  20 to 30 °C. Aspirate the supernatant. Gently vortex to dissociate the pellet.
  12. Resuspend cells in 3 mL of 1X PerFix-nc Buffer 3.
  13. Centrifuge the tube at 500 x g for 5 minutes at 20 to 30 °C. Aspirate the supernatant. Gently vortex to dissociate the pellet.
  14. Resuspend cells in 500 μL of PerFix-nc Buffer 3.
  15. 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 50 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 two drops of the positive VersaComp Antibody Capture Beads to the following compensation tubes:
    • CD39-PC5.5
    • FoxP3-Alexa Fluor 647
  3. Follow steps 2-15, skipping step 8, in the Sample Preparation procedure.
  4. 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-A 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.
  4. Create a CD3-APC-Alexa Fluor 750 vs. SSC-A dot plot and apply the Lymphs gate onto the plot. Draw a region to encompass the CD3+ T cells.
  5. Create a CD4-PC7 vs. CD3-APC-Alexa Fluor 750 dot plot and apply the CD3+ T cells gate onto the plot. Draw a region to encompass the CD3+/CD4+ T cells. These are the CD4+ T cells.
  6. Create a CD25-PE vs. FoxP3-Alexa Fluor 647 dot plot and apply the CD4+ T cells gate onto the plot. Draw a region around the FoxP3+ 25+ double positive population. These are the CD4+ FoxP3+ 25+ T-regulatory (T-reg) cells.
  7. Create a CD45RA-FITC vs. CD4-PC7 dot plot and apply the FoxP3+ 25+ T-reg gate onto the plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the 45RA- 4+ T-reg, 45RA+ 4+ T-reg, 45RA+ 4- T-reg and 45RA- 4- T-reg cell populations.
  8. Create a Helios-Pacific Blue (PB) vs. CD39-PC5.5 dot plot and apply the FoxP3+ 25+ T-reg gate onto the plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the Helios- 39+ T-reg, Helios+ 39+ T-reg, Helios+ 39- T-reg and Helios- 39- T-reg cell populations.
  9. Record the desired statistics.