DURAClone SC Hematopoietic 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 SC Hematopoietic Stem Cell Antibody Panel

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

  • CD4-FITC
  • CD4-PE
  • CD34-ECD
  • CD10-PC7
  • CD45-APC-Alexa Fluor 750
  • CD4-APC
  • 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

Sample Collection Tube

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: 425 – 470 nm and 530 – 570 nm
  • 488 nm: 505 – 790 nm
  • 633 nm: 650 – 790 nm

Sheath fluid

Flow cytometer calibration beads

Staining Procedure

SAMPLE PREPARATION 

NOTE: If the sample contains red blood cells, follow steps 3-4.  If not, these steps can be skipped.

  1. Add 100 μL of the sample to one tube of the DURAClone SC Mesenchymal Antibody Panel.

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

  3. Add 2 mL of VersaLyse. Vortex at high speed for 6-8 seconds and incubate for 10 minutes between 18 and 25  ̊C. Protect from light.

  4. Centrifuge the tube at 150 x g for 5 minutes; aspirate the supernatant.

  5. Add 3 mL of 1X PBS; vortex at high speed for 6-8 seconds.

  6. Centrifuge the tube at 150 x g for 5 minutes; aspirate the supernatant.

  7. Resuspend cells in 500 μL of 1X PBS/Fixative solution.

  8. 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 positive VersaComp Antibody Capture Beads to the following compensation tubes:
  3. Follow steps 2-8 in the Sample Preparation procedure.
  4. Follow standard procedures and instrument manufacturer instructions for compensation setup.

 

Analysis

  1. Create an FS INT vs FS TOF (time of flight) plot and leave the plot ungated. Draw a region/gate to encompass singlet events based on their low FS TOF values, thus excluding doublet events from further analysis.

  2. Create a FS INT vs SS INT plot and apply the singlets gate. Draw a region labelled as Scatter to encompass high forward scatter cells to exclude debris from further analysis.

  3. Create a CD45 vs SS INT plot and apply the Scatter gate. Draw a region labelled as CD45+ to encompass leukocytes.

  4. Create a CD34 vs SS INT plot and apply the CD45+ gate. Draw a region/gate to encompass the CD34+ cells containing HSCs (Hematopoietic Stem Cells) and HPCs (Hematopoietic Progenitor Cells).

  5. Create a CD45 vs SS INT plot and apply the CD34+ gate. Draw a region to encompass CD45dim clustered cells containing HSCs and HPCs.

  6. Create a FS INT vs SS INT plot and apply the CD45dim cluster gate. Draw a region to encompass HSCs/HPCs based on their low SSC, thus excluding apoptotic cells and platelet clumps.

  7. Create a CD45RA vs CD10 plot and apply the HSCs/HPCs gate. Draw 2 regions/gates to encompass CD10-CD45RA- cells and CD45RA+ cells, respectively.

  8. Create a CD133 vs CD10 plot and apply the CD45RA+ gate. Draw a quadrant region/gate encompassing CD133- CD10+ BLPs (B-cell Biased Lymphoid Progenitors), CD133+ CD10+ MLPs (Multi-Lymphoid Precursors), CD133+ CD10- GMPs (Granulocyte and Macrophage Progenitors) and LMPPs (Lymphoid- Primed Multi-Potent Progenitors), and CD133- CD10- late GMPs.

  9. Create a CD133 vs CD10 plot and apply the CD10- CD45RA- gate. Draw a quadrant region/gate to encompass CD133- CD10- EMPs (Erythro-Myeloid Progenitors) and CD133+ CD10+ populations MPPs (Multi-Potent Progenitors), respectively.

  10. Create a CD90 vs CD38 plot and apply the MPP gate. Draw a quadrant region/gate to encompass CD38dim CD90+ HSCs.

  11. Create a CD49f vs CD34 plot and apply the CD38dim CD90+ gate. Draw a region/gate to encompass CD133+ CD49f++