Q: You say manual counting of cells is eliminated when using your Cell Transformation Kits with Cell Recovery, but how can I quantify my cells and still recover them intact for further analysis?
A: All of our Cell Transformation Assays include a quantitation step that eliminates manual cell counting. However, this quantitaiton does require cell lysis. When using our Cell Recovery Compatible assays, it is recommended that duplicate wells (or more) be run for each sample. This will allow you to lyse and quantify cells in one well and then recover the cells from the duplicate well.
Q: Does this assay recover all cells or just colony forming cells?
A: Our 96-well cell transformation assay measures anchorage independent growth and includes the option to recover viable cells after incubation for re-culture. This kit will not separate colony forming cells from non-colony forming cells, however, most of the non-colony forming cells will die because they are typically anchorage-dependent and will not be viable without a solid surface to grow on. Even if some of the non-colony forming cells survive and remain viable, they will not grow during the assay incubation and their numbers will be negligible compared to colony forming cells.
Q: What is the composition of the 5X DMEM?
A: The DMEM provided with this kit is high glucose with L-glutamine.
Q: Can any media be used with this assay?
A: The DMEM is provided in the kit as a convenience, but any media that is compatible the cell line can be used, as long as it is at a 2X concentration.
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: 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 4ºC 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 many times do I need to pipette after adding the solubilization solution?
A: According to the protocol, the Matrix Solubilization Solution should be pipetted 10-12 times after adding to the wells to ensure complete solubilization. It is fine to pipette more than this if necessary, because this is a critical step and can result in a reduced and inaccurate signal if not thoroughly mixed. It is also important to mix well after adding the MTT Solution.
Q: How does the detection work?
A: Our fluorometric 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 assay #CBA-140, 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.
Our colorimetric soft agar assay uses MTT to colorimetrically detect live cells. MTT measures the activity of enzymes in living cells that reduce tetrazolium dye (yellow) to formzan (purple). The formzan is insoluble and requires addition of a detergent solution to dissolve the formzan into a purple solution that can be quantified between 500 and 600nm.
Q: Is it normal to form crystals after adding MTT?
A: MTT measures the activity of enzymes in living cells that reduce tetrazolium dye (yellow) to formzan (purple). The formzan is insoluble and requires addition of a detergent solution to dissolve the formzan into a purple solution that can be quantified between 500 and 600nm. The crystals you are observing are from the insoluble formzan and is normal.
Q: Will GFP interfere with the detection?
A: Cells that express GFP can be used with these assays because the GFP expression is negligible and won’t interfere with the signal of either assay. If you have a fluorescent plate reader, we recommend #CBA-140 because it has better sensitivity compared to #CBA-135.