DURAClone IM Dendritic Cells 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 Dendritic Cell Antibody Panel

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

  • CD4-FITC
  • CD4-PE
  • CD1c-PC5.5
  • CD11c-PC7
  • CD4-APC
  • CD123-APC-Alexa Fluor 700
  • 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.

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 200 μL of whole blood to one tube of the DURAClone IM Dendritic Cells 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 - 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 ready for 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 one drop of the positive VersaComp Antibody Capture Beads of the following compensation tubes:
  3. Follow steps 2-7 in the Sample Preparation procedure.
  4. Follow standard procedures and instrument manufacturer instructions for compensation setup.

Analysis

  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. 
  4. Create a 3-14-19-20-56 Lineage-PE vs. HLA-DR-Pacific Blue (PB) dot plot and apply the Leukocytes gate onto the plot. Draw a region to encompass the LINneg HLA-DR+ high population.
  5. Create a CD11c-PC7 vs. CD123-APC-Alexa Fluor 700 dot plot and apply the LINneg HLA-DR+ gate onto this plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CD11c- CD123+ Plasmacytoid dendritic cells (DCs) and the CD11c+ CD123- Myeloid DCs.
  6. Create a CD16-FITC vs. CD1c-PC5.5 dot plot and apply the Myeloid DCs gate from the CD11c-PC7 vs. CD123-APC-Alexa Fluor 700 dot plot onto this plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CD1c+ Myeloid DCs (MDC1) and the CD16+ Myeloid DCs.
  7. Create a CD16-FITC vs. Clec 9A-APC dot plot and apply the Myeloid DCs gate from the CD11c-PC7 vs. CD123-APC-Alexa Fluor 700 dot plot onto this plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the Clec 9A+ Myeloid DCs (MDC2).
  8. Record the desired statistics.