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. Improving upon the Invitrogen CyQUANT family of cell proliferation assays, we have developed the CyQUANT Direct Cell Proliferation Assay, with these benefits:
- More convenient workflow ideal for high-throughput screening—no washes, cell lysis, or temperature equilibrations required
- Measures independent of metabolic state of cells reducing risk for false positives
- Assess cell growth, cell viability, or compound toxicity in a range of applications
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 CyQUANTDirect 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.
With a dynamic range from less than 40 to more than 20,000 cells (approximately) depending on cell type and format, the CyQUANT Direct assay can be used in either 96, 384, or 1,536-well microplate formats and is compatible with most HTS and HCS readers (Figure 2).
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)
An accurate measurement of cytotoxicity and cell proliferation
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.
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 30 µL medium containing 10% FBS. Following incubation at 37°C for 48 hours with increasing concentrations of Tamoxifen, 30 µL 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.
CyQUANT Direct Assay references
Tediose T, Kolev M, Sivasankar B et al. (2010) Interplay between REST and nucleolin transcription factors: a key mechanism in the overexpression of genes upon increased phosphorylation. Nucleic Acids Res 38(9):2799–2812.
Hasan A, Pokeza N, Shaw L et al. (2011) The matricellular protein cysteine-rich protein 61 (CCN1/Cyr61) enhances physiological adaptation of retinal vessels and reduces pathological neovascularization associated with ischemic retinopathy. J Biol Chem 286(11):9542–9554.
Ian M. Dickerson and Edward B. Brown Methods of treating cancer using an agent that modulates activity of the calcitonin-gene related peptide ("CGRP") receptor. US. Patent Application 12/987,876, Aug. 4, 2011
For Research Use Only. Not for use in diagnostic procedures.