Nexcelom Bioscience

978-327-5340

Whole-Well Imaging for Accurate Counting of Non-Uniform Cell Populations

whole well image

Monitor Cell Growth and Proliferation using Label-Free Bright Field Imaging

Bright field images showing cell proliferation

Label-Free Bright Field Imaging to monitor cell proliferation

During a proliferation assay, the number of cells increased from 49323 cells on day 0 to 80621 cells on day 3 (curve on right)

Monitor cell proliferation over time

Monitor cell proliferation over time

Counting of Total and PI-Positive Suspension Cells at an Increasing Drug Concentration



Well D2 D4 D8
BF 87925 57718 39107
PI (+) 2654 7520 13171
Viability 96% 87% 66%

The number of PI-positive cells increased in a drug dose-dependent manner

Well D2

PI Positive Suspension Cells bright field image
PI Positive Suspension Cells fluorescent image

Well D4

PI Positive Suspension Cells bright field image
PI Positive Suspension Cells fluorescent image

Well D8

PI Positive Suspension Cells bright field image
PI Positive Suspension Cells fluorescent image
 
cell count increase

96-Well Based Cell Counting Assay for Microscale Screening Platform for CHO Cells

Assay protocol

  • Use optical quality 96-well plate
  • Make master dye containing Hoechst 33342 and propidium iodide
  • Load 200 µl of master dye mix per well
  • Add 3 µl of CHO cell suspension per well
  • Incubate 40 min at room temperature
  • Image 96-well plate on Celigo image cytometer (blue and red fluorescence channels)
  • Use “Direct Cell Counting” image analysis parameters for Hoechst+ and PI+ cells
  • Count all cells in each well
  • Obtain live cell density using live cell count: Hoechst+ - PI+
  • Obtain viability: (Hoechst+ - PI+)/Hoechst+
 

Direct Cell Counting using Image Analysis for Hoechst+ and PI+ Cells

CHO cell images

direct cell counting using Hoechst and PI

CHO cell images in the blue channel, red channel, merged image and counted cell image.

Experiment 1:

Prepared a dilution series of exponentially growing healthy CHO-S cells and examined viable cell density (VCD), coefficient of variation (CV) of VCD of healthy CHO-S cells

Comparison of viable cell density using a 96-well based Celigo method to one-sample based cell counting method.

comparison of viable cell density

Viable cell density is plotted against relative cell concentrations for the 96-well based and one-sample based method. The dashed black line from linear regression of 96-well based method (R2 = 0.999)

Comparison of CV of VCD using a 96-well based Celigo method to one-sample based cell counting method.


Comparison of CV of VCD using a 96-well

Coefficient of variation of VCD measurements is plotted against the measured cell concentration using a 96-well based and one-sample based method

Viability of does not change due to cell concentration in 96-well based and single sample based samples


Viability change due to cell concentration in 96-well

Percent viability is plotted against the measured cell concentration using the Celigo 96-well based and one-sample based method

Experiment 2:

Cells at two concentrations were treated with endoplasmic reticulum (ER) stress inducer tunicamycin to produce sample with lower viabilities

Cells treated with endoplasmic reticulum (ER) stress inducer tunicamycin

The viability and VCD decreased in a dose dependent manner for both 96-well based and one-sample based method.

Conclusion

96-well based method versus the one-sample based method showed excellent correlation in VCD, CV of VCD, and viability measurement. Based on the performed cell dilution series, 96-well based method has a counting range from 1x105 to 1x107 live cells/mL.

Citation:
Hansen HG, Nilsson CN, Lund AM, et.al. (2015) Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells. Scientific Reports 12 (5):18016

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