DURAClone IM B 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 IM B Cell Antibody Panel

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

  • CD4-FITC
  • CD4-PE
  • CD19-ECD
  • CD27-PC7
  • CD4-APC
  • CD38-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

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.

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 10 mL 1X PBS to 300 μL of whole blood. Centrifuge the tube at 300 x g for 10 minutes; aspirate the supernatant and resuspend the pellet in 10 mL of 1X PBS. Centrifuge the tube again at 300 x g for 5 minutes. Aspirate the supernatant and resuspend the pellet in 300 μL of 1X PBS.

  2. Add 100 μL of washed whole blood to one tube of the DURAClone IM B Cell Antibody Panel.

  3. Vortex at high speed for 6-8 seconds and incubate for 15 minutes between 20 and 30 °C. Protect from light.

  4. Add 2 mL of VersaLyse Solution, vortex at high speed for 1-3 seconds and incubate for 15 minutes between 20 and 30 °C. Protect from light.

  5. Centrifuge the tube at 200 x g for 5 minutes; aspirate the supernatant. Gently tap to dissociate the pellet.

  6. Add 3mL 1X PBS; centrifuge at 200 x g for  5 minutes. Aspirate the supernatant. Gently tap to dissociate the pellet. 

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

  8. The sample is ready for acquisition. Set the discriminator on the FS parameter such that the lymphocytes are not excluded from the 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. Follow steps 2-8 in the Sample Preparation procedure.  

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

Analysis 

  1. Create a CD45-Krome Orange vs. SSC dot plot and create a region to encompass the CD45+ leukocytes

  2. Create a CD19-ECD vs. SSC dot plot and create a region to encompass the CD19+ cells. The CD19+ cells are the B cells

  3. Create three dot plots and apply the CD19+ gate on all the three plots:
    • CD27-PC7 vs. IgD-FITC. Draw a quadrant to gate the IgD+ CD27- cell population; this is the naïve B cell population. Gate the IgD+ CD27+ population; these are the marginal zone B cells.

    • CD38-APC-Alexa Fluor 750 vs. CD21-PE. Draw a quadrant to delineate the CD21 low CD38 low cell population.

    • IgM-Pacific Blue vs. IgD-FITC. Draw a quadrant to delineate the IgD-IgM-, IgD+IgM- , IgD+IgM+, IgD-IgM+ cell populations.

  4. Create a CD38-APC-Alexa Fluor 750 vs. CD27-PC7 dot plot and apply the IgD-IgM- gate onto the plot. Draw a quadrant to delineate the following:

    • a. IgD-IgM- CD38-CD27+population. These are the class-switched memory B cells.

    • b. IgD-IgM- CD38-low CD27- population

    • c. IgD-IgM- CD38+ high CD27- population

    • d. IgD-IgM-CD38+ high CD27+ population. These are the plasmablasts.

  5. Create the following dot plots and apply the IgD+IgM+ gate onto them:

    • a. CD38-APC-Alexa Fluor 750 vs. CD27-PC7. Add a quadrant to delineate the following:

      • 1) IgM+CD27-CD38 dim population

      • 2) IgM+CD27+CD38- population: These are the class-unswitched memory B cells.

      • 3) IgM+CD27+ CD38+ high population

      • 4) IgM+ CD27-CD38- population.

    • b. CD24-APC vs. CD38-APC-Alexa Fluor 750 and draw a region to encompass the transitional B cells by applying the Boolean gate IgM-CD27-CD38 dim OR IgM-CD27- CD38 high.