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 

Example Data in Kaluza Analysis File (download)

  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. Draw a region around the CD45+/low SSC Lymphs cell population.
  4. Create a CD19-ECD vs. SSC-A dot plot and apply the Lymphs gate onto the plot. Draw a region to encompass the CD19+ B cells.
  5. Create the following dot plots and apply the CD19+ B cells gate onto these plots: 
    • Create an IgM-Pacific Blue (PB) vs. IgD-FITC dot plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the IgM- IgD+ B cells, IgM+ IgD+ B cells, IgM+ IgD- B cells and IgM- IgD- B cells.
    • Create a CD38-APC-Alexa Fluor 750 vs. CD21-PE dot plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CD38- CD21 high+ B cells, CD38+ CD21 high+ B cells, CD38+ CD21- B cells and CD38 low CD21 low B cells.
  6. Create a CD27-PC7 vs. IgD-FITC dot plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CD27- IgD+ Naïve B cells, CD27+ IgD+ Marginal zone B cells, CD27+ IgD- B cells and CD27- IgD- B cells. 
  7. Create a CD38-APC-Alexa Fluor 750 vs. CD27-PC7 dot plot and apply the IgM- IgD- gate from the IgM-Pacific Blue (PB) vs. IgD-FITC dot plot onto this plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the IgM- IgD- 38- 27+ Class switched memory B cells, IgM- IgD- 38+ high 27+ high Plasmablast cells, IgM- IgD- 38 high+ 27- B cells and IgM- IgD- 38 low 27 low B cells.
  8. Create another CD38-APC-Alexa Fluor 750 vs. CD27-PC7 dot plot and apply the IgM+ IgD+- gate from the IgM-Pacific Blue vs. IgD-FITC dot plot onto this plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the IgM+ IgD+ 38-  27+ Class unswitched memory B cells, IgM+ IgD+ 38 high+ 27+ B cells, IgM+ IgD+ 38 high+ 27- B cells and IgM+ IgD+ 38 dim 27- B cells.
  9. Create a CD24-APC vs. CD38-APC-Alexa Fluor 750 dot plot. Create the following boolean gate: "38 dim 27-" OR "38 high 27-”. Name the Boolean gate “IgM+ 38+ 27-” and apply the Boolean gate onto this plot. Draw a region to encompass the CD24+ CD38+ high population. These are the Transitional B cells.
  10. Record the desired statistics.