Assays for cell proliferation are widely used and many are available. We began using Invitrogen’s CyQUANT® NF Cell Proliferation Kit several months ago, when manual cell counting by hemocytometer became too time-consuming for the numbers of assays and replicates we were performing. The kit is invaluable for counting large numbers of cell samples.
This kit, which comes in sizes of 200 or 1000 reactions, includes CyQUANT® NF dye reagent, dye delivery reagent, and Hank’s Balanced Salt Solution (HBSS) concentrate. The dye is a readily cell-permeable, DNA-binding reagent, which is diluted in HBSS with or without the dye delivery reagent (which assists uptake), and is applied directly to cells in a 96-well plate after aspiration of growth media. After a 30-60 minute incubation, fluorescence is read using excitation at 485 nm and emission at 530 nm (similar to GFP). Fluorescence can be compared to a standard curve prepared simultaneously to estimate absolute cell number, or to a control condition to estimate relative proliferation.
We have used this system in siRNA-treated NIH-3T3, HeLa, and H1299 non-small-cell lung carcinoma cells with great success for comparing growth curves over a one- to two-week period. We seed 100 cells per well initially, and assay fluorescence 4 hours after plating to establish a baseline. Then, assays can be performed on replicate wells daily until overgrowth occurs. The reagent readily detects 100 cells, as advertised, but once cells near confluence, a linear fluorescence response is lost as they become clumped. We routinely use 50 uL of diluted reagent per well – half the recommended amount – so that the number of assays per kit is doubled. Including the dye delivery reagent has not been necessary for the cell types we have used. We measure fluorescence 30 minutes after dye addition, and have found that fluorescence is stable for at least another half-hour after that. Rather than using a microplate reader, as the manufacturer recommends, we scan the 96-well plate on a GE Healthcare Typhoon 9410 scanner, and quantify fluorescence intensity per well using ImageQuant software. Scanning the plate gives a visual readout of fluorescence, and allows manual exclusion of wells with clumped cells or other artefacts.
The speed and ease of use of the CyQUANT® NF assay is its greatest advantage over arduous manual cell counting or lengthy metabolic assays. Using half the recommended amount of reagent also makes this assay fairly cheap; only pennies per well. However, there are a couple of significant drawbacks. Firstly, we have found that well-to-well variability is high in our hands, so multiple replicates (we usually do six) are necessary to give acceptable standard deviations. Secondly, since the dye binds any DNA, it does not distinguish live from recently dead or apoptosing cells. For adherent cells, this is of minimal concern, since dead cells are usually sloughed off into the growth medium and removed, but this should be kept in mind when counting suspension cells. We have not tried counting non-adherent cells, but Invitrogen does include a protocol for this. Aside from these caveats, we have found CyQUANT® NF to be a handy cell-counting tool.