DURAClone IF T Helper Cell 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 Helper Cell Antibody Panel

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

  • CD4-FITC
  • IL4-PC7
  • CD4-APC
  • 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 sodium heparin 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, >755nm
  • 633 nnm: 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 200 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 Helper Cell 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 PerFix-nc Buffer 3.

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

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

  11. Sample is ready for acquisition.

SAMPLE PREPARATION FOR PERIPHERAL BLOOD MONONUCLEAR CELLS 

  1. Add 50 μL of activated PBMCs (containing  ~5 x105 cells) to a 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 200 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 Helper Cell 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 PerFix-nc Buffer 3.

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

  10. Resuspend cells in 500 μL of 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.

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:

    • IL4-PC7

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

  4. Follow steps 2-11 skipping step 6 in the Sample Preparation procedure for whole blood.

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

Analysis

  1. Create a CD3-Alexa Fluor 750 vs. CD4-APC dot plot. Create a region to encompass the CD3+CD4+ cells.

  2. On the CD4+CD3+ gated cells assess the cytokine production by:

    • Creating an IFNγ-FITC vs IL4-PC7 dot plot and a quadrant gate to differentiate subsets: IFNγ+ IL4-, IFNγ+ IL4+, IFNγ- IL4+ and IFNγ- IL4- cells.

    • Creating an IFNγ-FITC vs IL17a-Pacific Blue dot plot and a quadrant gate to differentiate subsets IFNγ+ IL17a-, IFNγ+ IL17a+, IFNγ- IL17a+ and IFNγ- IL17a- cells.