DURAClone SC Mesenchymal 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 Mesenchymal Cell Antibody Panel

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

  • CD4-FITC
  • CD4-PE
  • CD34-ECD
  • CD146-PC5.5
  • CD105-PC7
  • CD45-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

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: 430 – 470 nm and 530 – 570 nm
  • 488 nm: 560 – 600 nm
  • 633 nm: 715 – 735 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:

    • CD34-ECD
    • CD146-PC5.5
    • CD105-PC7

  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 ungated FS INT vs FS TOF dot plot. Add a region to encompass singlet events based on their low FS TOF values, thus excluding doublet events from further analysis.

  2. Create a CD45-APC-Alexa Fluor 750 vs CD34-ECD dot plot and apply the singlets gate . Create a region to encompass the CD45+ fraction to exclude leukocytes, the CD45- CD34- fraction, containing debris and pericytes, and the CD34+ fraction, containing endothelial progenitor cells containing MSCs.

  3. Create a SS vs CD14/19-Krome Orange dot plot and apply the singlets gate. Encompass CD14/CD19- cells containing MSCs.

  4. Create a CD90-FITC vs CD146-PC5.5 dot plot and apply CD45-CD34- gate. Encompass (e.g. by using a quadrant gate) the CD146+ cells to identify CD90+ and CD90- pericytes.

  5. Create a CD31-Pacific Blue vs CD146-PC5.5 dot plot and apply CD34+ gate. Create a CD31+CD146+ gate to identify endothelial progenitor cells and a CD31- CD146- gate to encompass the cellular fraction containing the MSCs.

  6. Create a CD73-PE vs CD90-FITC dot plot and apply the CD31-CD146- gate. Encompass the CD73+CD90+ fraction containing the MSCs.

  7. Create a CD105-PC7 vs CD90-FITC dot plot and apply CD73+CD90+ gate. Encompass the CD105-CD90+ fraction to identify the MSCs.

  8. Create SS vs CD14/CD19-Krome Orange dot plot and apply the MSCs gate. Create a gate linked to CD14/CD19-Krome Orange (created in step 3) to assess the portion of CD14- CD19- MSCs.

  9. Create a radar plot including axes for CD31, CD34, CD73, CD105, CD90, CD146 and apply a boolean CD45neg gate (“CD45-CD34-” OR “CD34+”) to verify isolated clustering of the MSCs.