////Suspension Cell Counting
Suspension Cell Counting2018-06-10T21:48:52+00:00

Suspension Cell Counting

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

Whole-Well Imaging for Counting of Non-Uniform Cell Populations
Ask a Specialist
Request a Quote
Celigo Product Guide
Applications Collection

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

Bright field images showing cell proliferation

Bright field images showing 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 manne

Well D2

Counting of Total and PI-Positive Suspension CellsCell counting propidium iodide

Well D4

Counting of Total and PI-Positive Suspension Cells 2

Well D8

Counting of Total and PI-Positive Suspension Cells 3Cell counting propidium iodide 3
celigo-cell-counting-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 CHO cell images

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)

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

CV density bar graph - one-sample based cell counting method

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

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

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

Percent viability comparison data

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 a sample with lower viabilities

One-sample vs 96-well viability

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 1×105 to 1×107 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