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      • Microfluidic Analysis
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      • Base Editing Platform
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      • Immune Cell Screening
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      T-SPOT.TB testing services.

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    • Clinical & Testing Services
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        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • Cellular and Humoral Immunoassays
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      T-SPOT.TB testing services.

      Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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    • Customization Services
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      • Radiosynthesis and Labeling
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        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      T-SPOT.TB testing services.

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    • Licensing
      • Gene Delivery Licensing
        CHOSOURCE expression platform

        Revvity's expression platform provides an enhanced system for the development and manufacturing of biotherapeutics that can be used in commercial manufacturing applications.

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      • Gene Expression Systems
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        CHOSOURCE expression platform

        Revvity's expression platform provides an enhanced system for the development and manufacturing of biotherapeutics that can be used in commercial manufacturing applications.

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      • Pin-point™ Base Editing Platform
        CHOSOURCE expression platform

        Revvity's expression platform provides an enhanced system for the development and manufacturing of biotherapeutics that can be used in commercial manufacturing applications.

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      • Virus Screening
        CHOSOURCE expression platform

        Revvity's expression platform provides an enhanced system for the development and manufacturing of biotherapeutics that can be used in commercial manufacturing applications.

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      CHOSOURCE expression platform

      Revvity's expression platform provides an enhanced system for the development and manufacturing of biotherapeutics that can be used in commercial manufacturing applications.

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    • Viral Vector Engineering and Manufacture
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        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      T-SPOT.TB testing services.

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      Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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    T-SPOT.TB testing services.

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  • Home
  • Knowledge Base
  • CellCountingSub Section
  • Comparing Manual and Automated Counting Methods
Cell Counting and Image Cytometry

Comparing Manual and Automated Counting Methods

Section
Cellometer Applications
Celigo Applications
Cell Counting Method Selection
Cell Counting and Image Cytometry FAQs
Cell-based Assays for Bioprocessing
Cell-based Assays for Gene Therapy Development
Cellometer Applications
Modern Virology Assays
Sub Section
Cell Counting with a Hemocytometer
Automated Analysis of Primary Cells
Brightfield Cell Analysis
Cell Analysis for Breweries and Biofuels
Cell Concentration and Viability for Blood-based Samples
Cell Counting with a Hemocytometer
Cell Viability for Clean and Messy Samples
Fluorescent Assays
Immuno-oncology Research Using Cellometer Instruments
Miscellaneous Cell Types
Topic
Comparing Manual and Automated Counting Methods
Comparing Manual and Automated Counting Methods
Counting Mammalian Cells Using a Hemocytometer
Counting Yeast Cells Using a Hemocytometer
History of the Hemocytometer
Sources of Hemocytometer Counting Errors
The Disposable Hemocytometer

Introduction

The Cellometer image cytometers are capable of performing automated concentration and viability measurement using both Trypan blue and fluorescent methods. The fluorescent Cellometer instruments, such as Auto 2000, K2 and Ascend, determine viability by counting cells stained with acridine orange (AO) and propidium iodide (PI). This allows for more reliable detection of nucleated cell in samples such as PBMCs and splenocytes that contain red blood cell contamination. The Cellometer Auto 2000 has been validated for reliability, reproducibility and linearity against manual counting using a hemocytometer following the recommended testing guidelines set by NIST (National Institute of Standards and Technology).

Cellometer Auto 2000 reliability and precision

Reliability of the Cellometer Auto 2000 counting method was determined by comparing the calculated % CV of two individuals. (Hemocytometer and Auto 2000). The precision of the Cellometer Auto 2000 counting method was determined by comparing the calculated % CV intra (single) user (Auto 2000). The reliability and precision tests used 5 µm diameter beads at a concentration of 1.0 x 106 beads/mL. Two users performed 6 counts each of the beads with a hemocytometer and 12 counts each of the beads on the Cellometer Auto 2000 under same conditions. The results obtained from the Cellometer Auto 2000 were compared with the results from the hemocytometer. Coefficients of variation (%CV) between each counting method and between users were calculated. The % CV was 2.9% for the reliability of the Cellometer Auto 2000 with the hemocytometer and between two users. Precision of the Cellometer Auto 2000 was 4.6% and 3.5% for % CV intra user 1 and user 2 respectfully and an % CV inter user 1 & 2 of 1.8%
 

Hemocytometer Bead Concentration (x106beads/mL) % CV intra
User 1 average (n=6) 1.05 4.9%
User 2 average (n=6) 1.11 6.9%
User 1 & 2 average (n=12) 1.08  
STDEV User 1 & 2 0.043  
%CV inter Uswer 1 & 2 4.0%  

 

Hemocytometer Bead Concentraion (x106beads/mL) % CV intra
Auto 2000 Bead Concentration (x106beads/mL) % CV intra
User 1 average (n=12) 1.01 4.6%
User 2 average (n=12) 1.04 3.5%
User 1 & 2 average (n=24) 1.03  
STDEV User 1 & 2 0.019  
%CV inter User 1 & 2 1.8%  
Hemocytometer & Auto 2000 Bead Concentration x106beads/mL) %CV intra
User 1 average (Hem & A2K) 1.03 2.4%
User 2 average (Hem & A2K) 1.08 3.5%
User 1 & 2 average (n=24) 1.05  
STDEV User 1 & 2 0.031  
%CV inter User 1 & 2 2.9%  

 

Cellometer Auto 2000 linearity

Following the guidelines published by NIST, linearity of the Auto 2000 was performed by measuring different Jurkat cell concentrations, plotted on a graph with expected cell concentrations compared with the measured cell concentrations. R2 value for the measure cell concentrations was >0.999 and the error bars overlaid with the expected cell concentration line.

Auto 2000 Serial dilution Jurkat cell concentrations
 

Auto-2000-serial-dilution-Jurkat-cell-concentrations


Error bars: +/- standard deviation for each average measured cell concentration
 

Dilution Expected Conc. Measured Conc. STDEV %CV
1 1.06E+07 1.06E+07 1.85E+05 1.8%
0.5 5.29E+06 5.50E+06 9.25E+04 1.7%
0.25 2.65E+06 2.69E+06 1.20E+05 4.5%
0.125 1.32E+06 1.28E+06 2.74E+04 2.1%
0.0625 6.61E+05 5.59E+05 3.78E+04 6.8%
0.03125 3.31E+05 2.79E+05 2.68E+04 9.6%
0.015625 1.65E+05 1.24E+05 1.21E+04 9.8%



For research use only. Not for use in diagnostic procedures.

ON THIS PAGE
  • Introduction
  • Cellometer Auto 2000 reliability and precision
  • Cellometer Auto 2000 linearity
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