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
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
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
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
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
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
