Using HydroFlex™ Platform for Gentle Washing of Cultured Cells in Microplates

Introduction

This article shows how the Tecan HydroFlex platform was effectively evaluated for gentle washing of weakly adherent P815 cells and the strongly adherent cell-line A431 in a 96-well format.

The new HydroFlex platform from Tecan has advanced features to suit a wide application range including vacuum filtration, washing of microplates, and magnetic bead separation.

When the HydroFlex platform is used for gentle cell washing, it provides individual control of important wash parameters, for instance speed settings for aspiration and dispense and wash head positioning. Fine-tuning of the wash protocol specific to the cell-type used, is also allowed by the HydroFlex platform.

Tecan’s HydroFlex™ platform

The wash head position relative to the rising liquid level in the wells is optimized by a very gentle drop-wise dispense mode together with a move function, so that cell detachment is minimized and good wash results are ensured, even in the case of weakly adherent cell types.

A HydroFlex platform, fitted with a standard wash head suited for ELISA and cell washing, was used for the cell-based assay described in this article.

When the HydroFlex platform is used for cell-based assays, the assay reactants can be removed with little or no effect on the viability of the used cells.

Experiments with semi-adherent and strongly adherent cells were carried out to determine the viability and wash efficiency of the cells. To visually confirm the gentle wash procedure, the cell layers were also observed before and after washing.

Material and methods

Instrument

Tecan HydroFlex platform fitted with the standard 8-way manifold and two inlet channels for wash buffer.

Microplates

Greiner Bio-One 96-well (Greiner Bio-One).

Reagents

Trypan Blue (TB), Dulbecco’s Modified Eagle’s Medium (DMEM, PAA Laboratories), 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT).

Assay protocol - Cell culture

Mouse mastocytoma cells (P815, ATCC-No. TIB-64) and human epidermoid carcinoma cells (A431, ATCC-No. CRL-1555) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 mM HEPES, 4 mM L-glutamine, 100 U ml-1 penicillin, 1 mM Na-pyruvate, 0.1 mg ml-1 streptomycin and 5% (v/v) fetal calf serum (FCS) (all from PAA-Laboratories, Linz, Austria), in a humid atmosphere at 7.5% CO2 and 37 °C.

A series of washing procedures spread over a period of four weeks were used to select adherent cells from the P815 cell line. Fresh DMEM supplemented (mentioned above) was used to replace the supernatant containing the suspension cells. For all assays, each well of a 96-well microplate was seeded with 2.104 cells of the respective cell line in 100 μl DMEM (supplemented as above).

The removal of a colored solution was monitored to determine the wash efficiency of the HydroFlex platform. Therefore, to start the washing procedure, the absorbance at 565 nm was determined (= 100 %) by adding 20 μl of 0.05 % Trypan Blue (TB) to each well. After washing, absorbance measurements were used to determine the remaining TB.

The MTT assay was carried out on cells with no washing treatment (= 100 % viability) and also on cells with a washing treatment (= x % viability) to determine cell viability.

The principle behind the MTT assay is that a viable and metabolically active cell reduces the soluble yellow tetrazolium salt MTT [3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide] by the mitochondrial dehydrogenase activity to form an insoluble dark formazan.

The viability and the number of viable cells can be determined by using the resulting absorbance values from the resolved formazan.

Wash programs

The wash programs listed below were tested for the effect of various wash volumes (600 μl in Program 1 and 800 μl Program 2) and the effect of repeated wash steps (one or two wash steps) on cell viability and wash efficiency.

 

Program 1

Wash 1x/2x

Z-position

10.5 mm + Move

Volume

600 μl

Head speed

1 mm/s

Wash rate

Drip mode

Aspirate

Mode

Normal

Z-position

8500 μm

Time

1 s

Head speed

1 mm/s

Aspiration rate

1

 

Program 2

Wash 1x/2x

Z-position

10.5 mm + Move

Volume

800 μl

Head speed

1 mm/s

Wash rate

Drip mode

Aspirate

Mode

Normal

Z-position

8500 μm

Time

1 s

Head speed

1 mm/s

Aspiration rate

1

All runs were respectively performed with A431 and P815 cells. For all assays, the wells of a 96-well microplate were seeded with 2 *104 cells of the respective cell line in 100 μl supplemented DMEM.

Results

Light microscopic images of the weakly adherent cell line P815 and the adherent cell line A431, before and after conducting Program 1 over above mentioned plates, are shown in Figures 1 and 2. There are no holes within the cell layer, as shown in the images.

These results prove that for gentle processing of weakly adherent cell lines and adherent cell lines, the HydroFlex platform provides a perfect solution and at the same time ensures intact cell layers after washing.

Figure 1. Weakly adherent cell line P815 before (left) and after washing (right) with PBS using Program 1

Figure 2. Adherent cell line A431 before (left) and after washing (right) with PBS using Program 1

The results of wash efficiency (TB-Test) and cell viability test (MTT test) after doing Program 1 or Program 2 with one or two washing steps is shown in Chart 1. The two wash programs were used to process two plates that contain A431 and P815 cells, respectively.

Chart 1. Absorbance values [%] of TB and MTT after wash procedure

μl

Program 1

Program 2

Wash

Wash

1x 600

2x 600

1x 800

2x 800

A431 Cells

Wash Efficiency [%]

Cell Viability [%]

 

95.6

98.3

97.3

98.6

81.0

84.1

76.1

75.96

 

P815 Cells

Wash Efficiency [%]

Cell Viability [%]

 

94.9

99.1

97.4

99.0

73.0

70.7

73.8

64.7

 

By using the HydroFlex platform, the alliance of high wash efficiency and a small affect on the cells is shown by photometric evaluation.

Chart 1 shows that recovered cell rate is over 75% with A431 cells and 70% with P815 cells. Due to minor adhesion which is often seen with cell assays, the recovery rates are marginally lower using Program 2 with 800 μl volume to wash P815 cells.

Figure 3. Content of TB and MTT after running Program 1 and Program 2 respectively

Using a one-step wash and aspirate program, a high washing efficiency of about 95% and cell viability of over 75% were obtained.

Conclusion

Excellent performance was demonstrated by the Tecan HydroFlex platform for automated washing of weakly adherent or adherent cells, together with efficient and gentle washing with good cell viability and very low detachment rates.

For many adherent and weakly adherent cell-types cultured in microplates, advanced control of vital wash parameters, such as wash head positioning as well as speed settings for aspiration and dispense, enable the wash conditions to be easily adjusted.

Acknowledgement

Tecan wishes to thank Mag. Tobias Kiesslich, Mag. Juergen Berlanda (Research Group Prof. B. Krammer), Department of Molecular Biology, University of Salzburg www.uni-salzburg.at/pdt

Literature

  1. Mosmann T.: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16; 65(1-2):55-63.

About Tecan

Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Jun 6, 2017 at 9:12 AM

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