Q: Is it acceptable to add antibiotic to the medium?
A: Antibiotics can be used in the media with this assay, and it will be able to diffuse through the cell agar layer. Any media that is regularly used can be used with this assay, as long as it is 2X.
Q: Does the media need to be replaced during the incubation?
A: The cells should be monitored during the 6-8 day incubation period and the media should be replaced if it is starting to turn yellow. The replacement media should be 1X.
Q: Can the prepared 1.2% agar solution and 2XDMEM/20%FBS be stored?
A: The prepared 1.2% agar solution and 2XDMEM/20%FBS can be stored at 4C as long as they remain sterile. The number of times the agar solution is heated should be minimized because this will alter the concentration.
Q: Can this assay be used for drug screening?
A: #CBA-150 was designed to be used for chemosensitivity testing; however, #CBA-130 can be used for this purpose as well. The advantage to using #CBA-130 is that it is more sensitive and is able to detect as few as 500 cells.
Q: How do I add a compound for testing?
A: When adding the 100 µL media overlay in section III, step 1 of the protocol (bottom of page 5), you would want to consider the volume dilution when figuring the concentration of any compound you add. You start with a 50 µL base layer and then add a 75 µL cell layer, so the total volume will be 225 µL. Therefore whatever compound you add during that step should be done at the appropriate concentration in order to end up at 1X final in 225 µL.
Q: Why are cells forming a monolayer on the bottom of the well?
A: If the cells are getting underneath the base layer and are growing on the bottom of the well, it suggests that the base layer is loose. You can avoid this by:
1. Adding the cell agar layer immediately after the base layer has solidified.
2. Add the cell layer gently, otherwise the force of the adding the cell layer can disrupt the tight interaction between the base layer and the wall of the well, allowing some cells to get underneath the base layer and therefore forming standard 2D culture.
Q: Can I pour the base layer and let it sit at 4ºC overnight?
A: We don’t recommend incubating the base layer at 4C for an extended time period because the gel will contract and create spaces around the well that the cells can fall down into. This will result in regular 2D growth rather than anchorage independent growth.
Q: How does the detection work?
A: Our Soft Agar Assays detect cells using CyQuant dye, which is a green fluorescent dye that shows a strong increase in fluorescence when bound to nucleic acids. In the Cell Transformation Assay, the dye is added after cell lysis and nucleic acid release, and is able to detect as few as 500 cells and can detect colonies before they are visible under the microscope. This low limit of detection allows for detection of fewer cells and thereby significantly reduces the incubation time. Using the traditional method, colonies need to be large enough to detect manually, which requires a longer incubation period.
Q: Will the CyQuant dye differentiate between living and dead cells?
A: CyQuant does not differentiate between living and dead cells. The assay starts with a fixed number of cells that is consistent between wells, which can be considered a background number. Cells that do not grow in soft agar or are inhibited by a compound would not be expected to produce a signal increase much above this level. Any signal above this background level can be considered cell growth. A cell dose curve can be run in parallel to identify cell number or the samples can be compared to each other for relative values.
Q: Can the cells be transfected while in soft agar?
A: It is not possible to perform the transfection when the cells are in the soft agar because this is a semi-solid matrix and the DNA will not be delivered to the cells. Our recommendation is to perform the transfection on cells in a standard dish and harvest the cells after 24 hours, at which time the DNA should be inside the cells. Cells can then be added to the soft agar and the incubation can be started.
Q: Can I freeze my samples before adding the CyQuant® dye?
A: Yes, the samples will be in lysis buffer and it is fine to freeze them until the detection step can be performed.
Q: What protocol do you recommend for staining the cells?
A: Although not part of the assay, colonies can be stained with INT for visualization. Colonies stained with INT cannot also be quantified with CyQuant. Here is a protocol for INT staining:
1. Prepare 0.1% p-iodonitro tetrazolium violet (INT) solution in 1X PBS. For example, add 100 mg of INT (Sigma I8377) to 100 mL of PBS.
2. Carefully aspirate culture medium on the top of the soft agar containing cell colonies, add INT staining solution (for a 24-well plate, 1 mL/well).
3. Incubate 16 hrs at 37ºC.
4. Before examining the colonies under microscope, replace the INT staining solution with PBS.
Q: How can the IC50 value be determined?
A: The IC50 can be calculated based on the results of CBA-130 by plotting RFU vs drug concentration, similar to Figure 2 in the product manual for #CBA-150.
Q: How do I know when to end the experiment?
A: Cell growth should be monitored under the microscope with our cell transformation assays. Even though there is a low limit of detection, you still want enough cells to give you reliable and consistent results. To determine the end point, you will look for an increase in cell number compared to when they were plated and a change in cell morphology, indicating colony formation. You will want to avoid too much cell growth or the cells will run out of room. Each cell line will have a different optimal incubation time, for example: most cancer cell lines need 7 days, very aggressive cells such as skin melanoma will only require 5 days, while for some slow growing brain tumor cells it may take 10 days.