Microscopy image of cells stained with a CyQUANT reagent.

The amount of DNA in each cell remains constant for a given cell line or cell type, so assays based on DNA content—like Invitrogen CyQUANT Cell Proliferation Assays—can be used to provide an accurate and simple measure of cell number. CyQUANT Cell Proliferation Assays are ideal for high-throughput screening, are more sensitive than colorimetric-based assays, and are not radioactive. Rapid and easy to use, CyQUANT Cell Proliferation Assays Kits require no washes, extractions, growth medium changes, or long incubations, and don't rely metabolic status of the cell. See your assay options with our Selection Guide.

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CyQUANT Assays Selection Guide

 CyQUANT Cell ProliferationCyQUANT NF (No Freeze) Cell Proliferation
Use
  • Sensitive, fluorescence-based method for quantifying cells and assessing cell proliferation and cytotoxicity
  • Microplate assay
  • Fast and sensitive fluorescence-based method for measuring proliferation end point assay
  • Microplate assay
Measurement
  • Total cell count of all live and dead cells
  • Cells must be lysed by freezing or with lysis buffer in order for stains to enter cells
  • Total cell count of all live and dead cells
Benefits of this assay
  • Cell samples can be frozen and stored for up to 4 weeks prior to assaying, allowing batch analyses of different time points
  • Broadest detection range
  • No lysis required for cell staining
  • Fastest incubation, 10 – 60 minutes
Linear detection range
  • 50 to 50,000 cells per well
  • Up to 250,000 with increased dye concentration
  • 100 to 20,000 cells per well
Ex/Em (nm)480/520 nm497/520 nm
Cat. No.C7026C35007


CyQUANT Cell Proliferation Workflow & Performance

Image of the 5 step CyQuant Cell Proliferation Assay workflow and the standard curve for fluorescence vs number of cells

Figure 1. Workflow for CyQUANT Cell Proliferation Assays multi-day tests or when you need precision. Quantitation of NIH 3T3 fibroblasts using the CyQUANT Cell Proliferation Assay Kit. Fluorescence measurements were made using a microplate reader with excitation at 485 nm and emission detection at 530 nm. The linear range of the assay under these conditions is from 50 to 50,000 cells per 200 µL sample. The inset shows the linearity that can be obtained at very low number of cells.

CyQUANT NF (No Freeze) Cell Proliferation Workflow & Performance

Image of the 4 step CyQuant NF Cell Proliferation Assay workflow and the standard curve for fluorescence vs number of cells

Figure 2. Workflow for CyQUANT NF Cell Proliferation Assays for when you need real-time data. Quantitation of CHO (M1WT3) cells using the CyQUANT NF Cell Proliferation Assay Kit. Fluorescence measurements were made using a microplate reader with excitation at 485 nm and emission detection at 530 nm. The linear range of the assay under these conditions is from 100 to 20,000 cells per 100 µL sample. The inset shows the linearity that can be obtained at very low number of cells.

 CyQUANT Direct Cell Proliferation Assays
Use
  • Fluorescence-based proliferation and cytotoxicity assay for microplate readers
  • Convenient workflow for high-throughput cytotoxicity and proliferation screening projects
Measurement
  • Only live, healthy cells stained
  • Masking dye blocks staining of dead cells or cells with compromised cell membranes
Benefits of this assay
  • No lysis step allows the assay to be easily multiplexed
  • Add-mix-read protocol make this ideal for HTS applications
  • 2 different fluorophore emission options available: 527 nm (green) and 645 nm (red)
  • Results correlate with metabolism-based assays
Linear detection range
  • 50 to 20,000 cells per well
Incubation & Assay time
  • 1 hour assay
Ex/Em (nm)508/527 nm (green)622/645 nm (red)
Cat. No.C35011C35013


CyQUANT DIRECT Cell Proliferation Workflow & Performance

CyQUANT Direct assay protocol

Figure 3. 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.

Figure 4. 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 5. 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.

Figure 6. 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

Total cell count
 CyQUANT Cell ProliferationCyQUANT NF (No Freeze) Cell Proliferation
Use
  • Sensitive, fluorescence-based method for quantifying cells and assessing cell proliferation and cytotoxicity
  • Microplate assay
  • Fast and sensitive fluorescence-based method for measuring proliferation end point assay
  • Microplate assay
Measurement
  • Total cell count of all live and dead cells
  • Cells must be lysed by freezing or with lysis buffer in order for stains to enter cells
  • Total cell count of all live and dead cells
Benefits of this assay
  • Cell samples can be frozen and stored for up to 4 weeks prior to assaying, allowing batch analyses of different time points
  • Broadest detection range
  • No lysis required for cell staining
  • Fastest incubation, 10 – 60 minutes
Linear detection range
  • 50 to 50,000 cells per well
  • Up to 250,000 with increased dye concentration
  • 100 to 20,000 cells per well
Ex/Em (nm)480/520 nm497/520 nm
Cat. No.C7026C35007


CyQUANT Cell Proliferation Workflow & Performance

Image of the 5 step CyQuant Cell Proliferation Assay workflow and the standard curve for fluorescence vs number of cells

Figure 1. Workflow for CyQUANT Cell Proliferation Assays multi-day tests or when you need precision. Quantitation of NIH 3T3 fibroblasts using the CyQUANT Cell Proliferation Assay Kit. Fluorescence measurements were made using a microplate reader with excitation at 485 nm and emission detection at 530 nm. The linear range of the assay under these conditions is from 50 to 50,000 cells per 200 µL sample. The inset shows the linearity that can be obtained at very low number of cells.

CyQUANT NF (No Freeze) Cell Proliferation Workflow & Performance

Image of the 4 step CyQuant NF Cell Proliferation Assay workflow and the standard curve for fluorescence vs number of cells

Figure 2. Workflow for CyQUANT NF Cell Proliferation Assays for when you need real-time data. Quantitation of CHO (M1WT3) cells using the CyQUANT NF Cell Proliferation Assay Kit. Fluorescence measurements were made using a microplate reader with excitation at 485 nm and emission detection at 530 nm. The linear range of the assay under these conditions is from 100 to 20,000 cells per 100 µL sample. The inset shows the linearity that can be obtained at very low number of cells.

Live cell count
 CyQUANT Direct Cell Proliferation Assays
Use
  • Fluorescence-based proliferation and cytotoxicity assay for microplate readers
  • Convenient workflow for high-throughput cytotoxicity and proliferation screening projects
Measurement
  • Only live, healthy cells stained
  • Masking dye blocks staining of dead cells or cells with compromised cell membranes
Benefits of this assay
  • No lysis step allows the assay to be easily multiplexed
  • Add-mix-read protocol make this ideal for HTS applications
  • 2 different fluorophore emission options available: 527 nm (green) and 645 nm (red)
  • Results correlate with metabolism-based assays
Linear detection range
  • 50 to 20,000 cells per well
Incubation & Assay time
  • 1 hour assay
Ex/Em (nm)508/527 nm (green)622/645 nm (red)
Cat. No.C35011C35013


CyQUANT DIRECT Cell Proliferation Workflow & Performance

CyQUANT Direct assay protocol

Figure 3. 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.

Figure 4. 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 5. 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.

Figure 6. 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

CyQUANT Protocols

Resources

Tools

Fluorescence SpectraViewer—This is an online tool for visualization of the excitation and emission of fluorescent reagents. This tool allows for checking spectral compatibility for multiple fluorophores.

Cell Viability, Proliferation and Cell Cycle Information—Find educational resources such as application notes, webinars, videos, articles, and more that cover the use of many of our reagents and kits for monitoring cell function.

BioProbes Journal of Cell Biology Applications—Stay up-to-date with highlights of the latest breakthroughs, and get information about new technologies and products.

Support

Cell Analysis Support Center—Find relevant technical information, tips and tricks, and answers to everyday problems.

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