Cell Transformation Assay—Tradition...

Introduction

Neoplastic transformation occurs via a series of genetic and epigenetic alterations that yield a cell

population that is capable of proliferating independently of both external and internal signals that

normally restrain growth.  For example, transformed cells show reduced requirements for extracellular

growth promoting factors, are not restricted by cell-cell contact, and are often immortal.  Anchorage-

independent growth is one of the hallmarks of transformation, which is considered the most accurate

and stringent in vitro assay for detecting malignant transformation of cells. 

Traditionally, the soft agar colony formation assay is a common method to monitor anchorage-

independent growth, which measures proliferation in a semisolid culture media after 3-4 weeks by

manual counting of colonies.  Standard soft agar assays are usually performed in 100-mm or 60 mm

dishes, where cells are allowed to grow inside a semisolid culture media for 3-4 weeks before sizable

colonies appear.  This method is quite cumbersome, time-consuming, and difficult when testing a large

number of samples.  Additionally, the manual counting of colonies is highly subjective; with varying

colony sizes, it’s difficult to determine meaningful results. 

Cell Biolabs CytoSelect™ 96-well Cell Transformation Assay does not involve subjective manual

counting of colonies or require a 3-4 week incubation period.   Instead cells are incubated only 6-8

days in a semisolid agar media before being solubilized, lysed and detected by the patented CyQuant®

GR Dye in a fluorescence plate reader （see Assay Principle below）.  This format provides a

quantitative, high-throughput method to accuray measure cell transformation.  Additionally, the

short incubation time （6-8 days） makes it possible to assay cells transiently transfected with oncogenes

or siRNA.   

The CytoSelect™ 96-well Cell Transformation Kit provides a robust system for screening oncogenes

and cell transformation inhibitors.  Each kit provides sufficient quantities to perform 96 tests in a

microtiter plate.

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Assay Principle

Related Products

1.   CBA-140: CytoSelect™ 96-Well Cell Transformation Assay （Cell Recovery, Fluorometric）

2.   CBA-135: CytoSelect™ 96-Well Cell Transformation Assay （Cell Recovery, Colorimetric）

3.   CBA-150: CytoSelect™ In Vitro Tumor Sensitivity Assay

4.   CBA-155: CytoSelect™ Clonogenic Tumor Cell Isolation Kit

5.   CBA-112: CytoSelect™ 96-Well Cell Invasion Assay （Basement Membrane, Fluorometric）

6.   CBA-106: CytoSelect™ 96-Well Cell Migration Assay （8µm, Fluorometric）

7.   CBA-106-C: CytoSelect™ 96-Well Cell Migration and Invasion Assay （8µm, Fluorometric）

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Kit Components

1.   CytoSelect™ Agar Powder （Part No. 113001）: One bottle – 1.2 g 

2.   5X DMEM Solution （Part No. 113002）: Two sterile tubes – 1.5 mL each 

3.   Agar Solubilization Solution （Part No. 113003）: One amber bottle – 6.0 mL

4.   8X Lysis Buffer （Part No. 113004）: One bottle – 3.0 mL

5.   CyQuant GR Dye （Part No. 10103）: One tube – 25 µL

Materials Not Supplied

1.   Cells and Culture Medium

2.   1X PBS

3.   37⁰C Incubator, 5% CO2 Atmosphere

4.   Light Microscope

5.   96-well Fluorometer

6.   Microwave or Heating Block

7.   Water bath

8.   （Optional） Positive Control cells such as NIH 3T3 （Ras G12V）

Storage

Store all components at 4ºC until their expiration dates.

Preparation of Reagents

•     1.2% Agar Solution: Place 1.2 g of Agar Powder in a sterile bottle, add 100 mL of sterile cell

culture grade water.  Microwave or boil until agar is compley dissolved.

•     2X DMEM/20% FBS Medium: In a sterile tube, dilute the provided 5X DMEM in sterile cell

culture grade water to 2X containing 20% FBS.  For example, to prepare a 5 mL solution, add 2

mL of 5X DMEM, 1 mL of FBS and 2 mL of sterile cell culture grade water.  Sterile filter the 2X

media to 0.2 µm.

•     CyQuant Working Solution: Immediay before use, prepare sufficient amount of the CyQuant

Working Solution by diluting the CyQuant GR Dye 1:400 with 1X PBS.  For example, add 10 µL

to 4 mL of 1X PBS.  Use the solution immediay; do not store the CyQuant Working Solution.

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Assay Protocol （must be under sterile conditions）

I. Preparation of Base Agar Layer

1.   Melt 1.2% Agar Solution in a microwave and cool to 37⁰C in a water bath.

2.   Warm 2X DMEM/20% FBS medium to 37⁰C in a water bath.  Allow at least 30 minutes for

the temperature to equilibrate.

3.   Mix equal volumes of 1.2% Agar Solution and 2X DMEM/20% FBS medium in a sterile, pre-

warmed tube by inverting several times.  Immediay transfer 50 µL of the mixture to each

well of a 96-well sterile flat-bottom microplate.  Gently tap the plate a few times to allow the

agar solution to evenly cover the wells. 

Note: 1） Work quickly with the agar solution to avoid gelation.  Also, try to avoid adding air

bubbles to the well.  2） To avoid fast and uneven evaporation that leads to aberrant results,

we suggest not using the wells on the plate edge, or filling the edge wells with medium to

reduce evaporation.

4.   Transfer the plate to 4⁰C for 30 minutes to allow the base agar layer to solidify. 

5.   Prior to adding the Cell Agar Layer （Section II）, allow the plate to warm up for 15 minutes at

37⁰C.

II. Preparation of Cell Agar Layer （samples should be assayed in triplicate）

1.   Melt 1.2% Agar Solution in a microwave and cool to 37⁰C in a water bath.

2.   Warm 2X DMEM/20% FBS medium to 37⁰C in a water bath.  Allow at least 30 minutes for

the temperature to equilibrate.

3.   Harvest and resuspend cells in culture medium at 0.4 - 4 x 10⁵ cells/mL, keep the cell

suspension warm in a 37⁰C water bath.

4.   Mix equal volumes of 1.2% Agar Solution, 2X DMEM/20% FBS media, and cell suspension

（1:1:1） in a sterile, pre-warmed tube by inverting several times.  Immediay transfer 75 µL of

the mixture to each well of the 96-well flat-bottom microplate already containing the solidified

base agar layer （25 µL of cell suspension containing 1000-10000 cells/well will be seeded）.

Note:  Work quickly with the agar solution to avoid gelation, but gently pipette as not to

disrupt the base layer integrity.  Also, try to avoid adding air bubbles to the well.   Always

include negative control wells that contain no cells in the cell agar layer.

5.   Transfer the plate to 4⁰C for 15 minutes to allow the cell agar layer to solidify. 

III. Quantitation of Anchorage-Independent Growth

1.   Add 100 µL of culture medium containing cell growth activator（s） or inhibitor（s） to each well. 

2.   Incubate the cells for 6-8 days at 37⁰C and 5% CO₂.  Examine the cell colony formation under

a light microscope.

3.   Remove culture medium by inverting the plate and blotting on paper towel.  Gently tap several

times.

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4.   Add 50 µL of Agar Solubilization Solution to each well of the 96-well plate.  Incubate for 1 hr

at 37⁰C.

5.   Pipette each well 5-10 times to ensure complete agar solubilization.

6.   Add 25 µL of 8X Lysis Buffer to each well.  Pipette each well 5-10 times to ensure a

homogeneous mixture.

7.   Incubate the plate at room temperature for 15 minutes.   

8.   Transfer 10 µL of the mixture to a 96-well plate suitable for fluorescence measurement. 

9.   Add 90 µL of the CyQuant Working Solution to each well.  Incubate 10 minutes at room

temperature.  

10. Read the plate in a 96-well fluorometer using a 485/520 nm filter set. 

Cell Dose Curve （optional）

1.   Harvest and resuspend cells in culture medium at 1 - 5 x 10⁶ cells/mL.

2.   Prepare a serial of 2-fold dilution with culture medium, including a medium blank.

3.   Transfer 125 µL of each cell dilution to a microfuge tube.  Add 50 µL of Agar Solubilization

Solution and 25 µL of 8X Lysis Buffer to each tube.  Vortex each tube to ensure a

homogeneous mixture.  Incubate the tubes at room temperature for 15 minutes.   

4.   Transfer 10 µL of the mixture to a 96-well plate suitable for fluorescence measurement. 

5.   Add 90 µL of the CyQuant Working Solution to each well.  Incubate 10 minutes at room

temperature.  

6.   Read the plate in a 96-well fluorometer using a 485/520 nm filter set. 

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Example of Results

The following figures demonstrate typical results with the CytoSelect™ 96-well Cell Transformation

Assay Kit.  Fluorescence measurement was performed on SpectraMax Gemini XS Fluorometer

（Molecular Devices） with a 485/538 nm filter set and 530 nm cutoff.  One should use the data below

for reference only.  This data should not be used to interpret actual results.

50

40

30

20

10

0

0

1000

2000       3000       4000

50

40

30

20

10

0

0       5000 10000 15000 20000 25000

Cells/mL （x1000）

Cells

Figure 1.  HeLa Cell Dose Curve.  Cervical carcinoma HeLa cells were resuspended at 4 x 10⁶

cells/mL and titrated 1:2 in culture medium, followed by addition of Agar Solubilization Solution,

Lysis Buffer, and Cyquant® GR Dye detection （as described in the Cell Dose Section）.  Results were

shown by cell concentration or by actual cell number in CyQuant Detection. 

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15

10

5

0

HeLa

20000 10000 5000 2500 1250                     625        313

Cells Seeded/Well

NIH3T3

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Figure 2. Anchorage-Independent Growth of HeLa Cells.  HeLa cells were seeded at various

concentrations and cultured for 6 days.  HeLa cell transformation is determined according to the assay

protocol.

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Figure 3. HeLa Colony Formation.  HeLa cells were cultured for 14 days according to the assay

protocol.  Colonies were visualized by 0.1% p-iodonitro tetrazolium violet （INT） staining. 

Calculation of Anchorage-Independent Growth

1.   Compare RFU values with the Cell Dose Curve and extrapolate the cell concentration in soft

agar.

2.   Calculate the Total Transformed Cell Number/Well 

Total Transformed Cells/Well = cells/mL in soft agar x 25 µL/125 µL x 0.125 mL/well 

For example: If you extrapolate your RFU value from your cell dose curve and determine

you have 500,000 cells/mL in your soft agar sample.

Total Transformed Cells/Well = 500,000 cells/mL x 0.2 x 0.125 mL/well = 12,500 cells/well

References

1.   Shin SI, Freedman VH, Risser R, and Pollack R. （1975） Proc Natl Acad Sci U S A. 72:4435-9.

2.   Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW and Weinberg RA. （1999）

Nature 400:464-8.

License Information

CyQuant® GR Dye is licensed from Molecular Probes （Invitrogen）.

Warranty

These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in

accordance with their instructions.  THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED

WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR

WARRANTY OF FITNESS FOR PARTICULAR PURPOSE.  CELL BIOLABS’ sole obligation and purchaser’s

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