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)
- 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).
- Add 2 mL of VersaLyse, vortex on high for 1-3 seconds and incubate for 15 minutes at laboratory conditions (18-25°C).
- The sample is ready for acquisition.
- For sample acquisition on Navios/Gallios:
- 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.
- Acquire the stained tube prepared using DuraClone IM Count Tube.
Note: Keep the FL2 channel on and set PMT voltage at minimum setting
- Create an FSC-A vs. FSC-H dot plot and draw a region to encompass the singlets cell population and exclude the doublets.
- 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.
- 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.
- 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.
- 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.
- 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.
- Record the desired statistics of all the gated cell populations. It is recommended to acquire 3000 bead events for estimating cell counts.
- Use the total number of beads events from the tube/pouch label or Certificate of Analysis to calculate cells/uL.