Protocol C00162 DuraClone IM Count

This protocol is for reference purposes only.  It may be necessary to adapt the method for specific research needs.

SAMPLE PREPARATION FOR WHOLE BLOOD (Example)

  1. Pipette 100 µL of whole blood into DuraClone IM Count Tube (Normal blood contains ~3000-11700 white blood cells per µL). Vortex at high speed for 6-8 seconds and incubate for 15 minutes at laboratory conditions (18-25°C).
  2. Add 2 mL of VersaLyse, vortex on high for 1-3 seconds and incubate for 15 minutes at laboratory conditions (18-25°C).
  3. The sample is ready for acquisition.
  4. For sample acquisition on Navios/Gallios:
  1. Prepare an unstained sample by lysing 100µL of whole blood as per step(2) and use it to set the PMT voltages for FL1 and FL4 channels by setting the X-median of the unstained sample at 0.3 for each channel.
  2. Acquire the stained tube prepared using DuraClone IM Count Tube.

Note: Keep the FL2 channel on and set PMT voltage at minimum setting

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-FITC 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 7-AAD vs. SSC-A dot plot and apply the Leukocytes gate onto this plot. Draw a region to encompass the 7-AAD+ cells. These are the DEAD leukocyte cells. Draw a region to encompass the 7-AAD- population. These are the LIVE leukocytes.  
  5. Create a CD45-FITC vs. FL2-A dot plot. Create a region around the events that show high fluorescence for FITC and FL2-A. This are the Beads.  
  6. Create a histogram plot for CD45-FITC and apply the Beads gate onto this plot. Select and apply a Linear gate onto this plot and adjust the linear gate to encompass the Bead population and exclude any aggregates. These are the bead singlets.
  7. Record the desired statistics of all the gated cell populations. It is recommended to acquire 3000 bead events for estimating cell counts.
  8. Use the total number of beads events from the tube/pouch label or Certificate of Analysis to calculate cells/uL.