Celigo-based cell-counting and viability assay for mammalian cells

doi:10.1038/protex.2016.038

Method Article

Celigo-based cell-counting and viability assay for mammalian cells

https://doi.org/10.1038/protex.2016.038

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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.

Cell biology

Biological techniques

VCD

viability

mammalian cells

CHO

high-throughput

Celigo

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 pipetting

Add 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 well

Incubate 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.

I declare no conflict of interest.

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Celigo-based cell-counting and viability assay for mammalian cells

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