This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Celigo-based cell-counting and viability assay for mammalian cells
Henning Gram Hansen
CHO Cell Line Engineering and Design, NNF-CfB, Technical University of Denmark
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Accurate assays for counting mammalian cells are important tools when performing experiments in cell biology. In general, commercially available cell-counting assays suffer from low throughput and high costs of consumables. Here, we describe a cost-efficient Hoechst and propidium iodide (PI)-staining protocol that accurately counts the number of viable mammalian cells in 96-well microplates based on image cytometry on a Celígo® Image Cytometer. Hands-on time for one microplate is less than 20 minutes and the entire procedure including incubation takes approximately 1 hour. Since all steps prior to image cytometry analysis involve liquid handling and since the Celígo® Image Cytometer is automation-friendly, this protocol has the potential to be integrated into automated liquid handling workflows.
Keywords
VCD, viability, mammalian cells, CHO, high-throughput, Celigo
Figure 1
96-Well Greiner™ 655090 Plate
1000xPI (sterile-filtered 0.4 mg/mL propidium iodide in dH2O, Sigma, cat# P4170)
2500xHoechst (‘Hoechst 33342’ 12.3 mg/mL 20 mM, ThermoFischer Scientific, cat# 62249)
CD CHO Medium (ThermoFischer Scientific, cat# 10743029)
200 mM L-Glutamine (Lonza, cat# 17-605)
CHO-S suspension cells - the protocol can probably also be used for other mammalian cells (adherent and suspension cells)
Celígo® S Cell Cytometer (Nexcelom)
Make the staining solution:
o 8 µM Hoechst, 0.4 µg/mL PI in complete media (CD CHO + 8 mM L-Glutamine)
Pipet 3 uL cell suspension to wells in the 96-well plate (a multi-channel pipette can be used)
o Remember to resuspend cells before pipettingAdd 200 uL staining solution to each well (a multi-channel pipette can be used)
Pipet 150 uL up and down three times in each well (a multi-channel pipette can be used)
o Aspirate at a steady pace
o Dispense at a relatively fast pace to ensure proper mixing
o Perform each pipetting step at different spots on the bottom of the wellIncubate at room temperature in the dark for 40 min to allow the cells to sediment and for the Hoechst stain to increase in intensity
o Hoechst and PI stains are stable for at least two hours.-Scan wells on the Celigo using the mask (blue channel; Hoechst) and target 1 (red channel; PI) application.
o Use fixed exposure time and gain settings for the red channel.Use a fixed gate that divide PI positive and negative cells
o Make sure to have healthy cells \(wt/parental/host cell line) in one well so you know that your PI-gate is dividing healthy and dead cells correctly. Notes:
Equations:
Viability="PI-negative cells in the well"/"total cells in the well"
VCD="total cells in the well"1000/3Viability
Working range for healthy as well as stressed/dying (long-term cultures) cells:
2e5-2e6 total cells/mL
The wide linear dynamic range in the paper only applies to healthy cells.
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Posted 20 Jun, 2016
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Associated Publications
Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
by Henning Gram Hansen, Claes Nymand Nilsson, Anne Mathilde Lund, +7
Scientific Reports (21 March, 2016)
Method Article
Celigo-based cell-counting and viability assay for mammalian cells
Henning Gram Hansen
CHO Cell Line Engineering and Design, NNF-CfB, Technical University of Denmark
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 20 Jun, 2016
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Accurate assays for counting mammalian cells are important tools when performing experiments in cell biology. In general, commercially available cell-counting assays suffer from low throughput and high costs of consumables. Here, we describe a cost-efficient Hoechst and propidium iodide (PI)-staining protocol that accurately counts the number of viable mammalian cells in 96-well microplates based on image cytometry on a Celígo® Image Cytometer. Hands-on time for one microplate is less than 20 minutes and the entire procedure including incubation takes approximately 1 hour. Since all steps prior to image cytometry analysis involve liquid handling and since the Celígo® Image Cytometer is automation-friendly, this protocol has the potential to be integrated into automated liquid handling workflows.
Figure 1
96-Well Greiner™ 655090 Plate
1000xPI (sterile-filtered 0.4 mg/mL propidium iodide in dH2O, Sigma, cat# P4170)
2500xHoechst (‘Hoechst 33342’ 12.3 mg/mL 20 mM, ThermoFischer Scientific, cat# 62249)
CD CHO Medium (ThermoFischer Scientific, cat# 10743029)
200 mM L-Glutamine (Lonza, cat# 17-605)
CHO-S suspension cells - the protocol can probably also be used for other mammalian cells (adherent and suspension cells)
Celígo® S Cell Cytometer (Nexcelom)
Make the staining solution:
o 8 µM Hoechst, 0.4 µg/mL PI in complete media (CD CHO + 8 mM L-Glutamine)
Pipet 3 uL cell suspension to wells in the 96-well plate (a multi-channel pipette can be used)
o Remember to resuspend cells before pipettingAdd 200 uL staining solution to each well (a multi-channel pipette can be used)
Pipet 150 uL up and down three times in each well (a multi-channel pipette can be used)
o Aspirate at a steady pace
o Dispense at a relatively fast pace to ensure proper mixing
o Perform each pipetting step at different spots on the bottom of the wellIncubate at room temperature in the dark for 40 min to allow the cells to sediment and for the Hoechst stain to increase in intensity
o Hoechst and PI stains are stable for at least two hours.-Scan wells on the Celigo using the mask (blue channel; Hoechst) and target 1 (red channel; PI) application.
o Use fixed exposure time and gain settings for the red channel.Use a fixed gate that divide PI positive and negative cells
o Make sure to have healthy cells \(wt/parental/host cell line) in one well so you know that your PI-gate is dividing healthy and dead cells correctly. Notes:
Equations:
Viability="PI-negative cells in the well"/"total cells in the well"
VCD="total cells in the well"1000/3Viability
Working range for healthy as well as stressed/dying (long-term cultures) cells:
2e5-2e6 total cells/mL
The wide linear dynamic range in the paper only applies to healthy cells.
Associated Publications
Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells
by Henning Gram Hansen, Claes Nymand Nilsson, Anne Mathilde Lund, +7
Scientific Reports (21 March, 2016)