DURAClone IF T Cell Activation 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 IF T Cell Activation Antibody Panel

3 compensation kits, each kit containing six single color tubes:

  • CD4-FITC
  • CD4-PE
  • IL2-PC7
  • CD8-Alexa Fluor 700
  • CD3-Alexa Fluor 750
  • CD4-Pacific Blue

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

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
  • 488 nm: 504 – 545 nm, 560 – 600 nm, >755 nm
  • 633 nm: 715 – 735 nm and >755 nm

Sheath fluid

Flow cytometer calibration beads

Staining Procedure

SAMPLE PREPARATION FOR WHOLE BLOOD 

  1. Add 50 μL of activated blood to an appropriately labeled test tube.

  2. Add 25 μL of PerFix-nc Buffer 1. Vortex until red pellet is dissociated. Incubate 15 minutes at room temperature.

  3. Add 2 mL 1X PBS. Vortex and centrifuge at 150 x g for 5 minutes. Aspirate the supernatant.

  4. Add 25 μL of Fetal Bovine Serum. Vortex to resuspend the cells.

  5. Add 300 μL of PerFix-nc Buffer 2. Vortex.

  6. Transfer the entire contents into one tube of the DURAClone IF T Activation Antibody Panel.

  7. Vortex the tubes at high speed for 6-8 seconds. Incubate 45 minutes at room temperature. Protect from light.

  8. Add 3 mL of 1X PerFix-nc Buffer 3.

  9. Vortex and centrifuge 500 x g for 5 minutes. Aspirate the supernatant.

  10. Resuspend cells in 500 μL of 1X PerFix-nc Buffer 3.

  11. 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. 

 

SAMPLE PREPARATION FOR PERIPHERAL BLOOD MONONUCLEAR CELLS

  1. Add 50 μL of activated PBMCs (containing  ~3 x105 cells) to an appropriately labeled test tube.

  2. Add 25 μL of PerFix-nc Buffer 1. Vortex until pellet is dissociated. Incubate 15 minutes at room temperature.

  3. Add 2 mL 1X PBS. Vortex and centrifuge at 150 x g for 5 minutes. Aspirate the supernatant.

  4. Add 25 μL of Fetal Bovine Serum. Vortex to resuspend the pellet.

  5. Add 300 μL of PerFix-nc Buffer 2. Vortex.

  6. Transfer the entire contents into one tube of the DURAClone IF T Activation Antibody Panel. 

  7. Vortex the tubes at high speed for 6-8 seconds. Incubate 45 minutes at room temperature. Protect from light.

  8. Add 3 mL of 1X PerFix-nc Buffer 3.

  9. Vortex and centrifuge 500 x g for 5 minutes. Aspirate the supernatant.

  10. Add 500 μL of 1X PerFix-nc Buffer 3. Vortex.

  11. 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 the same pouch.

  2. Add one drop of the positive VersaComp Antibody Capture Beads to the following compensation tubes:

    • IL2-PC7 compensation tube.

  3. Vortex at high speed for 6-8 seconds and incubate for 15 minutes between 18 to 25 ºC. Protect from light.

  4. Follow steps 2-11 skipping step 6, in the Sample Preparation procedure.

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

 

Analysis 

  1. Create a FS INT vs FS TOF dot plot and a singlet gate to eliminate any doublets (identified by higher values on FS TOF)

  2. Create a CD3-Alexa Fluor 750 vs. SSC dot plot and apply the singlets gate. Create a region to encompass the CD3+ cells.

  3. Create a CD4-Pacific Blue vs. CD8-Alexa Fluor 700 dot plot and apply the CD3+ gate. Create two regions to encompass the CD4+ and CD8+ cells individually.

  4. On the CD4+ and CD8+ gated cells assess the cytokine production by:

Creating an IFNγ-FITC vs TNFα-PE dot plot and a quadrant gate to differentiate subsets: IFNγ+ TNFα-, IFNγ+ TNFα+, IFNγ- TNFα+ and IFNγ- TNFα- cells.

Creating an IFNγ-FITC vs IL2-PC7 dot plot and a quadrant gate to differentiate subsets IFNγ+ IL2-, IFNγ+ IL2+, IFNγ- IL2+ and IFNγ- IL2- cells.