I am trying to use the CyQUANT Direct Cell Proliferation Assay Kit (from Invitrogen) to detect proliferation in human pulmonary aorta smooth muscle cells (HPASMCs) and human pulmonary aorta endothelial cells (HPAECs). The kit is a fluorescence based protocol (em 480/ ex 535). I have been using the SMGM-2 and EGM-2 from Lonza. I am having trouble with high background fluorescence. For example, a blank with only assay buffer and media reads ~7145 and a plate containing cells reads between 7280 and up. I have the plate reader set to read from the bottom of the plate (I got better results this way) and I have compared clear and black plates (with transparent bottom) and I saw no difference. I also tried using media with no phenol and that didn't significantly alter the background readings (only down to 7087). I have just been trying to include a blank so I can subtract out the difference, but I can't always do that.
My question is: Has anyone worked with this kit before? If so, did you have trouble with background readings and what steps did you take to reduce the background?
Thank you!
I use this reagent extensively. While I have not been able to reduce background, I have found that the background readings vary widely depending on the plate reader. While there will always be some measurable background, the Envision and Enspire plate readers from Perkin Elmer, and the BioTek Synergy HT, and BioTek Synergy Neo plate readers are able to give linear results down to 50 cells per well of a 96 or 384-well plate. I have found that some other plate readers do not have enough sensitivity for an acceptable dynamic range of readings for CyQUANT, so you may want to try other readers if they are available. Also, you always need to use a blank (well with no cells, but with reagent added) and subtract this background value from all other readings. In my experience, CyQUANT readings are essentially meaningless without the background subtraction step.
Best of luck!
-Nate
Maybe your excitation/emission is incorrect? I believe it's best at 480/520.
I switched to the MultiTox-Glo Cytotoxicity Assay kit from Promega. It work very well and the data is consistent and reproducible. I also use BrdU. Together these two assays are great to measure cell proliferation and cell viability.
I am having the same problem with this kit. The background saturates everything, inclusive the blank wells with no cells. I also tried different cells (just in case it was a specific problem of my cell line), different templates, media with no phenol, different reader set up.... I have asked the company support service but none of their suggestions has worked.
It is a pity to find so apparently easy protocols, spend time and money and don't get any result.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence