Protocol

Harvesting Cells Under Anchorage-Independent Cell Transformation Conditions for Biochemical Analyses

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Science's STKE  30 Apr 2002:
Vol. 2002, Issue 130, pp. pl7
DOI: 10.1126/stke.2002.130.pl7

Abstract

Most molecular biology and biochemical analyses use cultured cells grown in anchorage-dependent monolayer conditions. The standard oncogenic transformation assay for cell lines is usually performed in soft agar rather than in monolayers because of the higher transformation efficiency of cells in soft agar. However, cells suspended in soft agar cannot be readily recovered for studying inducible biochemical and molecular events. We developed an over-agar assay that enables us to study tumor promoter-induced cell transformation and the associated biochemical or molecular events under anchorage-independent conditions.

Introduction

Most molecular biology or biochemistry studies use cultured cells grown in anchorage-dependent (AD) monolayer conditions. However, the standard oncogenic transformation assay for cell lines is performed in soft agar rather than in monolayers because of the higher transformation efficiency of cells in soft agar. Because recovering cells from semi-solid soft agar involves plucking individual cells or colonies with a pipette, this method is not useful for studies of inducible biochemical and molecular events.

To overcome this limitation, we developed an over-agar assay that enables us to study tumor promoter-induced cell transformation and the associated biochemical or molecular events under anchorage-independent (AI) conditions (Fig. 1). This assay was developed for the JB6 mouse epidermal cell system derived from genetically varied mice, a model that has been used extensively for studying the genetic susceptibility to neoplastic transformation, promotion, and progression (1,-2, reviewed in 3). This model is also a well-developed cell culture system for studying tumor promotion, inhibition of tumor promotion, and reversion phenotypes in vitro (1-17). The JB6 cells are available in tumor promotion-sensitive (P+), promotion-resistant (P), or transformed (Tx) variants. These variants are a series of cell lines representing "earlier-to-later" stages of preneoplastic (P → P+) to neoplastic (P+ → Tx) progression (3-7). In P+ cells, tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), tumor necrosis factor-α (TNF-α), and arsenic, stimulate the formation of large, tumorigenic, AI colonies in soft agar at a high frequency. In contrast, the P cells form colonies in soft agar at a frequency about 1% that of P+ cells, and the colonies are considerably smaller (1-9).

Fig. 1.

A schematic of the over-agar assay procedure.

The over-agar culture method described here has been used to compare the activation of the transcription factor activating protein-1 (AP-1) in response to tumor promoters under AD and AI conditions (18). This method was also used to prepare JB6 P+ and P cells for mRNA differential display analysis (19-20). The method is simple and can be adapted for use with other cell lines that are assayed for AI growth in soft agar.

Materials

Cell Culture Reagents and Supplies

JB6 P+ Clone 41 or Clone 41.5a cells (ATCC, Manassas, VA), or available from Nancy H. Colburn, NCI, Frederick, MD

Fetal bovine serum (FBS) (Gemini Bio-Product, Calabasas, CA), heat inactivate at 56°C for 30 min

Trypsin, 2.5%, porcine parvovirus and mycoplasma tested (Invitrogen)

L-Glutamine, 100× solution, cell culture grade (Invitrogen)

Gentamicin (Quality Biological, Gaithersburg, MD)

Basal minimum medium (2× BME) (Invitrogen)

Eagle's minimum essential medium (EMEM) (BioWhittaker, Walkersville, MD)

T-25 (25 cm2), T-75 (75 cm2) flasks (Corning)

Petri dishes, 60 mm (Corning)

37°C humidified 5% CO2 incubator

Chemicals

12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma-Aldrich)

Epidermal growth factor (EGF) (Collaborative Product, Bedford, MA)

Dimethylsulfoxide (DMSO), silylation grade (Pierce, Rockford, IL)

Difco Batch Agar (BD Biosciences)

Other Materials

15- and 50-ml sterile screw-cap tubes

Tabletop centrifuge for 15- and 50-ml screw-cap tubes

37°C, 40°C, and 44°C water baths

100-ml glass bottles with caps

Recipes

Recipe 1: Ca2+, Mg2+-free phosphate-buffered saline (PBS)
Na2HPO4•2H2O1.44 g
KH2PO41 g
NaCl8 g
KCl0.2 g
Dissolve in 1 liter of H2O and autoclave or sterile filter.
Note: 1× PBS solution without Ca2+ or Mg2+ can be purchased from BioWhittaker, Wallkersville, MD.
Recipe 2: Growth Medium
Heat-inactivated FBSVolume of FBSVolume of PBS (Recipe 1)Volume of 2× BME
2%2 ml18 ml40 ml
5%5 ml15 ml40 ml
10%10 ml10 ml40 ml
20%20 ml0 ml40 ml
For the concentration of FBS desired, add the indicated amounts of FBS and PBS to the 2× BME.
Note: A 10% FBS growth medium solution is most frequently used in the JB6 cell transformation assay.
Recipe 3: TPA Stock Solution
Prepare a 10-μg/ml solution by dissolving 0.5 mg of TPA in 50 ml of DMSO. Store in 50-μl aliquots at –20° to –70°C.
Recipe 4: EGF Stock Solution
Prepare a 10-μg/ml solution by dissolving 0.5 mg of EGF in 50 ml of sterilized H2O. Store in 50-μl aliquots at –20° to –70°C.
Recipe 5: 0.03% Trypsin
Dilute 2.5% trypsin to 0.03% with Ca2+, Mg2+-free PBS (Recipe 1). Store at 4°C.
Recipe 6: JB6 Cell Growth Medium
EMEM
Heat-inactivated FBS5%
L-Glutamine2 mM
Gentamicin25 μg/ml

Instructions

In the following procedure, the first step is the preparation of the 0.5% Agar Growth Medium solution that is spread evenly over the bottom of a petri dish. Next, the cells are added in suspension over this layer and treated with a phorbol ester or growth factors to induce transformation. After a period in culture, the cells can be collected and analyzed for biochemical and molecular events associated with neoplastic transformation. All the procedures are performed under sterile conditions. If a large number of cells is needed for analysis, the procedures may be scaled up (for example, 20 ml of 0.5% Agar Growth Medium may be used in 150-mm petri dishes).

Preparation of 1.25% Agar

1. Pre-warm fifty 50-ml autoclave-safe glass bottles in a 40°C water bath.

2. Heat 1800 ml of deionized H2O in a 4-liter flask with 2-liter calibration to 80° to 90°C on a hot plate. Use a glass petri cap to prevent evaporation.

3. . Add 25 g of dry Difco Batch Agar to the flask containing the 80° to 90°C water. Stir with a glass rod until lumps disappear. While the flask is still being heated, adjust the volume to 2000 ml with H2O. Stir until thoroughly mixed.

4. Add 40 ml of the 1.25% agar solution to each of the 100-ml bottles previously held at 40°C. Cap bottles loosely.

5. Autoclave for 15 min on slow exhaust. Allow bottles to cool for a few minutes then tighten caps. Store at room temperature or at 4°C.

Preparation of the 0.5% Agar Growth Medium Dishes

The directions provided below are for 100 ml of 0.5% Agar Growth Medium--enough to prepare about 14 60-mm dishes. Volumes may be adjusted for larger sized dishes as needed.

1. Prepare the Growth Medium (Recipe 2) at the desired concentration of FBS, and warm in a 44°C water bath.

2. Melt 40 ml of 1.25% agar in a microwave oven until just molten, swirling intermittently. Remove the bottle as soon as the agar solution becomes clear.

3. Cool the 1.25% agar at room temperature for 2 to 3 min.

4. Place the bottle containing the agar in the 44°C water bath.

5. When both the Growth Medium and the 1.25% agar have reached 44°C, mix the 1.25% agar with the Growth Medium to make a 0.5% Agar Growth Medium solution.

6. Pipette 6 ml of the 0.5% Agar Growth Medium mixture into each sterile 60-mm petri dish. Avoid forming bubbles and spread the mixture evenly by slowly rotating the dish.

Note: Reagents such as TPA (Recipe 3) or EGF (Recipe 4) should be added to the 0.5% Agar Growth Medium just before pipetting it into the petri dishes. Typically, a final concentration of 10 ng/ml TPA or EGF is used. Control plates to which the solvent (DMSO or water) for the TPA or EGF is added should also be prepared.

7. Allow the 0.5% Agar Growth Medium layer to harden for 30 to 40 min at room temperature in a sterile laminar flow hood.

8. Store the dishes overnight in a 37°C incubator or seal in a bag and store at 4°C. The dishes can be stored this way for several days.

Note: Refrigerated dishes should be warmed to 37°C before use.

Preparation of Cell Suspension and Performing the Over-Agar Assay

1. Two or three days before the experiment, plate the JB6 cells in a screw-cap T-25 or T-75 flask in a final volume of 10 to 15 ml of JB6 Cell Growth Medium (Recipe 6).

2 Grow the cells in a 37°C, 5% CO2 humidified incubator until the cells are 80 to 90% confluent.

3. Wash the monolayer of cells in T-25 flask or T-75 flask twice with 1 to 2 ml of Ca2+, Mg2+-free PBS (Recipe 1), aspirating the PBS after each wash. .

4. Add 1 ml of 0.03% Trypsin (Recipe 5).

5. Incubate in a 37°C, 5% CO2 humidified incubator for 3 to 6 min or until 90% of the cells are detached.

6. Add 1 ml of EMEM containing 10% heat-inactivated FBS (EMEM-FBS).

7. Gently resuspend cells with a sterile pipette three or four times to make a single-cell suspension.

Note: Observe a sample of the suspension with a microscope to confirm that it consists of single cells rather than clumps.

8. Determine the concentration of cells and adjust the suspension to 0.33 × 104 cells per ml in EMEM-FBS.

Note: If the cells are to be plated on the TPA- or EGF-containing 0.5% Agar Growth Medium, then TPA or EGF should be added to the cell suspension just before transferring them onto the plates. The final concentration of TPA or EGF should be 10 ng/ml.

9. Transfer 3 ml of the cell suspension (containing a total of 1 × 104 cells) to the 0.5% Agar Growth Medium plate and incubate at 37°C, 5% CO2.

10. Culture the cells on the 0.5% Agar Growth Medium plate for 14 to 21 days, during which time colonies of transformed cells will form in the TPA- or EGF-treated dishes.

11. Count the number of cells in each colony and the number of colonies using a microscope.

Note: Each colony should contain more than eight cells.

Harvesting the Cells

1. Transfer the liquid growth medium and any unattached cells to a 15-ml conical tube.

2. Wash the agar surface gently with 3 to 4 ml of EMEM-FBS or PBS to dislodge the remaining cells and add these cells to the tube containing the growth medium and unattached cells.

3. Centrifuge the recovered cells at 1000g for 3 min at room temperature.

4. Analyze the cell pellet for biochemical or molecular events associated with transformation.

Note: Cells can be harvested at any time during the transformation process.

Troubleshooting

High Background

The colonies grown in the control group (without added TPA or EGF) should be between zero and 100 colonies per 104 cells plated. Cells that have been passaged many times can demonstrate high rates of spontaneous transformation. Therefore, cells with low passage numbers should be used or the concentration of serum in the growth medium can be decreased to 8% or less to reduce the likelihood of spontaneous transformation.

Low Transformation Efficiency

Low transformation efficiency may result from poor-quality serum, TPA, or EGF. We recommend testing different batches or lots of these reagents with the standard soft agar assay if this is a problem. Another alternative is to increase the serum concentration to 15 to 20%.

Notes and Remarks

The guiding principle in developing an over-agar assay for any given cell line is that the efficiency of AI growth should be comparable to that in soft agar. The advantage of the over-agar assay over the standard soft agar assay is that the cells are easily recoverable. If conditions for growth in the soft agar assay have been optimized, then start the over-agar culture with the same conditions used for the standard soft agar assay. The volume and concentration of the cell suspension to layer over the agar can be changed. Key variables in the optimization of either soft agar or over-agar AI growth assays include cell density (cells/plate), type and source of agar, growth medium, serum, and other supplements. Additionally, the concentration of the transforming agents and serum can be varied to suit other cell types.

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