CytoSelect™ 96-Well Cell Transformation Assay (Soft Agar Colony Formation)

1. CytoSelect™ Agar Powder : One 0.24 g tube
2. 5X DMEM Solution : One 1.5 mL sterile tube
3. Agar Solubilization Solution : One 1.5 mL amber tube
4. 8X Lysis Buffer : One 1 mL tube
5. CyQuant GR Dye : One 10 μL tube

1. Cells and Culture Medium
2. 1X PBS
3. 37 °C 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)

• Uses traditional 3D soft agar matrix
• Fully quantify cell transformation with no manual cell counting
• Results in 7-8 days, not 3 weeks

• 1.2 % Agar Solution: Place 0.24 g of Agar Powder in a sterile bottle, then add 20 mL of sterile cell culture grade water. Microwave or boil until agar is completely 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 2.5 mL solution, add 1 mL of 5X DMEM, 0.5 mL of FBS and 1 mL of sterile cell culture grade water. Sterile filter the 2X media to 0.2 μm. Note: You may substitute your own medium in place of the DMEM we provide, but ensure that it is at a 2X concentration.
• CyQuant Working Solution: Immediately 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 immediately, do not store the CyQuant Working Solution. 4

I. Preparation of Base Agar Layer

1. Melt 1.2 % Agar Solution in a microwave and cool to 37 °C in a water bath.
2. Warm 2X DMEM/20 % FBS medium to 37 °C 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. Immediately 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. Notes: • Work quickly with the agar solution to avoid gelation. Also, try to avoid adding air bubbles to the well. • 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 4 °C 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 37 °C.

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

1. Melt 1.2 % Agar Solution in a microwave and cool to 37 °C in a water bath.
2. Warm 2X DMEM/20 % FBS medium to 37 °C 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 37 °C 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. Immediately 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 4 °C 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 37 °C and 5 % CO2. Examine the cell colony formation under a light microscope. 5
3. Remove culture medium by inverting the plate and blotting on paper towel. Gently tap several times.
4. Add 50 μL of Agar Solubilization Solution to each well of the 96-well plate. Incubate for 1 hr at 37 °C.
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 2-fold serial 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. 6

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 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 0.125 mL/well = 62,500 cells/well