CyQUANT® Direct Cell Proliferation Assay
Metabolic activity is influenced by factors other than treatment, which can include cell type, confluence, media conditions and additives, temperature, and passage number. Compounds may also interfere with the enzymes that generate the metabolically based readout. For these reasons, a metabolic-based assay may not be ideal for some studies of cytotoxicity and proliferation.
CyQUANT® Direct Cell Proliferation Assay - An Ideal Assay for High-throughput Screening
The CyQUANT® Direct Cell Proliferation Assay kit consists of two components: a green fluorescent nucleic acid stain and a background suppression dye. Both components can be stored at room temperature for quick and convenient use. The nucleic acid dye is a live cell-permeable reagent that mainly concentrates in the nucleus of mammalian cells. The suppression dye is impermeable in live cells and suppresses “green” fluorescence, eliminating the need to perform wash steps (Figure 1). The combination of these two components results in an assay based on both DNA content and membrane integrity.
|Figure 1. CyQUANT® Direct assay protocol. The CyQuant Direct assay is a homogenous, lysis-free cell proliferation and cytotoxicity assay designed for use with multi-well plates (96, 384 or 1,536 plate formats), making it ideal for high-throughput screening applications. The reagent is added directly to cells in complete medium, incubated for 30 to 60 min and read on standard plate readers.|
The fluorescence intensity plateaus within 30 to 60 minutes after reagent addition and the signal remains stable for more than 7 hours offering workflow convenience and robustness in projects with large sample numbers (Figure 3).
Figure 2. Linearity of CyQUANT® Direct assay signal. CHO cells were plated at densities of 0 - 20,000 per well in a 384-well microplate. Cells were labeled with CyQUANT® Direct according to the microtiter plate assay format protocol. Fluorescence intensities, measured with a fluorescence microplate reader using FITC filter set, varied linearly with respect to cell number of the range of approximately 40 to 20,000 cells. The inset shows the measurement range from 0 - 1,250 cells per well. The results shown represent averages of eight experiments per data point.
Figure 3. Stability of CyQUANT® Direct assay signal. CyQUANT® Direct 2x detection reagent was added to adherent CHO cells in serum-containing medium, and fluorescence was read from 5 minutes to 7.5 hours after reagent addition. Fluorescence signal intensity reached a plateau within 30 - 60 minutes of reagent addition, and remained stable for more than 7 hours.
The CyQUANT® Direct assay is based on a cell-permeant DNA-binding dye in combination with a background suppression reagent. As DNA content is highly regulated, cell number estimates are very accurate. The masking dye blocks staining of dead cells and cells with compromised cell membranes, causing only healthy cells to be stained. Therefore, the CyQUANT® Direct assay measures proliferation as well as cytotoxicity. The concordance of the CyQUANT® Direct assay with cytotoxicity assays based on cell metabolism or energy states is excellent (Figure 4). Moreover, recent studies using diverse sets of cytotoxic compounds and assay types have shown that cell number (proliferation) is among the most sensitive indicators of cytotoxicity. 1-3
|Figure 4. Cytotoxicity measurements using the CyQUANT® Direct assay. Measurements of cytotoxicity differences across different cell types were performed using the CyQUANT® Direct assay. Hep-G2, Jurkat, human aortic smooth muscle cells (HASMC), and human pulmonary aortic smooth muscle cells (HPASMC) were seeded in 384-well plates at a density of 5,000 cells per well with 30µl medium containing 10% FBS. Following incubation at 37 degrees celcius for 48 hours with increasing concentrations of Tamoxifen, 30µl of CyQUANT® Direct reagent was added into each well. Fluorescence measurements were made after 60 minutes. Fluorescence intensities were normalized to DMSO alone treatment, and IC50 curves were generated. The results shown represent averages of readings from eight wells per data point. As shown in the figure, the two primary cell types (HASMC and HPASMC) were significantly more sensitive to Tamoxifen than two transformed cell lines, adherent Hep-G2, and suspension Jurkat cells.|
Rama Nadella*, Joe B. Blumer†, Guangfu Jia*, Michelle Kwon*, Talha Akbulut‡, Feng Qian§, Filip Sedlic‡, Tetsuro Wakatsuki‡, William E. Sweeney Jr.‖¶, Patricia D. Wilson‖¶, Stephen M. Lanier† and Frank Park*‡ Activator of G protein signaling 3 promotes epithelial cell proliferation in PKD JASN August 1, 2010, Volume 21, Issue 8, pages 1275-1280