Ficoll separation is routinely used to isolate mononuclear cells from bone marrow, peripheral blood, and umbilical cord blood. Monocytes and lymphocytes form a buffy coat under a layer of plasma, where the cells are collected, then washed. Typically, some residual platelets and red blood cells are mixed in with the mononuclear cells. The degree of platelet and red blood cell contamination can vary significantly between donors and operators. The Cellometer Auto 2000 Cell Viability Counter utilizes dual fluorescence counting to generate accurate live cell and dead cell counts with no interference from red blood cells and platelets.

Experimental Procedure

  1. Combine 20µl of PBMC sample and 20µl of AO/PI dye mixture and mix well by pipetting up and down
  2. Load 20µl of sample into the disposable counting chamber
  3. Allow cells to settle in chamber for less than 1 minute
  4. Insert chamber into Cellometer instrument
  5. Select assay from menu
  6. Enter sample ID manually or scan in with bar code reader
  7. Preview bright-field cell image and adjust focus (if necessary)
  8. Click “Count” to begin analysis
  9. Review images and counting results on-screen
  10. Images, cell count, concentration, % viability, and cell diameter data are saved to a secure network location

Accurate Cell Count and Viability Results

Each PBMC sample is incubated with an acridine orange / propidium iodide (AO/PI) dye mixture. The AO dye stains DNA in the cell nucleus of both live and dead cells. Mature mammalian red bloods cells don’t contain nuclei, so only peripheral blood mononuclear cells are stained, for a more accurate total PBMC count. Propidium iodide DNA-binding dye is used to determine cell viability. Healthy cells are impermeable to the PI dye. Only dead (non-viable) cells with compromised membranes are stained. Cells stained with both AO and PI fluoresce red due to Förster resonance energy transfer (FRET).

  • Live nucleated cells are counted in the green channel
  • Dead nucleated cells are counted in the red channel
  • There is no interference from platelets or red blood cells

PBMC Bright field image

PBMC Dual fluorescence image

In less than 60 seconds, the Auto 2000 reports live cell count and concentration, dead cell count and concentration, mean cell diameter, and % viability for the sample.  Bright field and fluorescent cell images can be viewed, saved, and exported for presentation or publication. Download the full Nexcelom PBMC Application Note for more information.