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Cell Counting Tutorials

Automated Concentration and Viability Measurement of PBMC using the Cellometer® Vision

Issues related to the current PBMC manual counting method

  • Potential judgment error due to residual red blood cells
  • Sample loading errors, such as too much liquid, air bubbles, or wear-out hemactyometer
  • Operator fatigue due to high background in the PBMC sample, such as platelets
  • Calculation error during data conversion after counting
  • Potential contamination during washing and cleaning
Cellometer Vision Counting Method

1. Prepare PBMC sample following the standard protocol
2. Mix 20 µl of PBMCs with 20 µl of fluorescence dye staining solution
3. Mix well
4. Load 20 µl of stained PBMC sample into Cellometer® slides

Assay Protocol:  PBMC Cellometer Images
Linearity and Consistency for PBMC Concentration Measurement
Linearity for PBMC Concentration Measurement

Results

  • Viable and nonviable PBMCs are specifically stained with AOEB
  • PBMCs dilution series showed linearity of R2 = 0.9991
  • Consistently measure PBMC from concentration of 2 X 105 to 2 X 107 cells/ml
  • %CV calculated ranges from 3.5% to 14.1%, which are in acceptable range
Determine PBMC Viability

Results

  • PBMCs sample stored in 4°C condition for 1-7 days
  • Viability of PBMCs reduced by ~40% after one week incubation
  • Measurement stays consistent in various concentrations
Comparison to Hemacytometer and Flow Cytometer

Results

  • PBMC count compared between hemacytometer, flow cytometer and Cellometer®
  • Cellometer® showed results closest to expected value
  • Hemacytometer showed results with a P = 0.00001
  • Concentration: cells/ml
  • Total nucleated cells measured by fluorescence (green + red).
  • All cells measure by bright field only
Specifically identify PBMC from RBC Contamination samples

Conclusions

  • Cellometer Vision provides a rapid and cost-effective method for concentration and viability measurement of PBMCs
  • The method uses very small amount of sample: less than 20 micro-liter
  • Residual red blood cell concentrations do not influence PBMC concentration and viability measurements
  • The method has a large linearity and good consistency

Viewing: Automated Concentration and Viability Measurement of PBMC using the Cellometer® Vision

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