CyQUANT Cell Proliferation Assays

Cell proliferation analyses play a pivotal role in studying cell growth and differentiation, offering insights into compound toxicity and tumor cell growth inhibition crucial for drug development. Cell proliferation assays leverage the sensitivity of fluorescent dyes to help provide accurate and reliable measurements, offering insights into cell viability, proliferation rates, and cytotoxicity.

CyQUANT cell proliferation assays are a versatile tool for researchers using a microplate format to evaluate cell growth and proliferation dynamics. Common measurements for these cell proliferation assays include DNA content or DNA synthesis. CyQUANT cell proliferation assays are particularly well-suited for high-throughput screening, boasting superior sensitivity compared to traditional colorimetric assays. These assays offer rapid and user-friendly protocols, eliminating the need for washes, extractions, or long incubations, enabling accurate results without dependence on the cell's metabolic status. Explore our array of easy-to-use CyQUANT cell proliferation assays for microplates.

CyQUANT cell proliferation selection guide

	CyQUANT Cell Proliferation	CyQUANT NF (No Freeze) Cell Proliferation	CyQUANT Direct Cell Proliferation Assays	CyQUANT Direct Red Cell Proliferation Assay
Use	Sensitive, fluorescence-based method for quantifying cells and assessing cell proliferation	Fast and sensitive fluorescence-based method for measuring proliferation end point assay	Fluorescence-based proliferation and cytotoxicity assay for microplate readers Convenient no-wash workflow for high-throughput screening
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	Only live, healthy cells stained Masking dye blocks staining of dead cells or cells with compromised cell membranes
Benefits	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	No lysis step allows the assay to be easily multiplexed Add-mix-read protocol makes this ideal for HTS applications Results correlate with metabolism-based assays
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	50 to 20,000 cells per well
Incubation & assay time	Multi-day assay	1 hour assay	1 hour assay
Ex/Em (nm)	480/520 nm	497/520 nm	508/527 nm (green)	622/645 nm (red)
User guide	CyQUANT Cell Proliferation Assay Kit	CyQUANT NF Cell Proliferation Assay Kit	CyQUANT Direct Cell Proliferation Assay Kit	CyQUANT Direct Red Cell Proliferation Assay Kit

Interested in measuring new DNA synthesis? Click-iT EdU Cell Proliferation Assay for microplates

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Cell proliferation assays for microplates

Measuring DNA content

Measuring DNA content is a crucial aspect of cell proliferation assays, and CyQUANT Cell Proliferation Assays offer an accurate microplate-based fluorescence method quantifying DNA content. Because the amount of DNA in each cell remains constant for a specific cell line or type, CyQUANT cell proliferation assays utilize this highly regulated and constant amount of cellular DNA to enable a precise measure of cell number. The CyQUANT assay can be used at multiple time points to calculate the average proliferation rate of a cell population. Binding of the CyQUANT dye to DNA is independent of metabolic state, so signal windows and fluorescence intensities can be compared across a range of conditions and cell types.

A variety of CyQUANT assay formats provide options for different workflows, including multi-day and endpoint assays for total cell numbers and assays for live cells.

Learn more about workflow and performance of CyQUANT cell proliferation assays below

Measuring new DNA synthesis

Various assays assess cell proliferation by measuring the rate of new DNA synthesis in cells. These assays involve incorporating nucleoside analogs, such as EdU or BrdU, into newly synthesized DNA. After incorporation, detection methods like click chemistry or immunostaining are used to quantify the extent of new DNA synthesis. These assays enable valuable insights into cell proliferation dynamics and can be tailored to specific experimental needs based on factors like throughput, sensitivity, and compatibility with fluorescence-based readouts.

The Click-iT Plus EdU Microplate Assay is optimized for microplate fluorescence analysis:

HTS compatible—Simple and rapid workflow
Multiplex ability—Compatible with a range of fluorescence multiplex options
Signal enhancement—Amplex UltraRed dye–amplified signal (568/585, ex/em)

Learn more about Click-iT EdU Microplate Cell Proliferation Assay

Figure 1. Click-iT EdU proliferation assay for microplates

Workflow and performance of CyQUANT cell proliferation assays

CyQUANT cell proliferation assay

CyQUANT Cell Proliferation Assays offer a reliable and sensitive method for quantifying cell proliferation based on cellular DNA content in a microplate format. The proprietary dye CyQUANT GR enhances fluorescence when bound to nucleic acids, allowing for the accurate measurement of cell proliferation rates by assessing DNA content within a broad linear detection range (Figure 2, 3). CyQUANT cell proliferation assays are known for their reliability in studying cell proliferation dynamics and their high sensitivity in detecting changes in cellular growth. Additionally, these assays can be easily tailored to specific experimental needs and are compatible with fluorescence-based readouts, making them versatile options for a wide range of cell proliferation studies.

diagram of cell proliferation assay workflow

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Figure 2. Workflow for CyQUANT Cell Proliferation assay multi-day tests. The CyQUANT Cell Proliferation assay enables comparison of cell samples from different time points. Simply remove culture media and freeze to store samples for up to 4 weeks, then thaw, add the CyQUANT GR dye in lysis buffer, incubate, and measure the fluorescence on a microplate reader.

Figure 3. 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 graph shows the linearity that can be obtained at very low number of cells.

graph of fluorescence vs number of cells

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CyQUANT NF (No Freeze) cell proliferation assay

The CyQUANT NF (no freeze) Cell Proliferation Assay offers a rapid and sensitive microplate-based method for quantifying cell proliferation without requiring cell freezing or prolonged incubations. This assay combines a cell-permeant DNA-binding dye with a membrane permeabilization reagent (Figure 4). This quantitation method uses fluorescence measurements with an excitation wavelength of 485 nm and emission detection at 530 nm, allowing for accurate determination of cell proliferation rates (Figure 5). CyQUANT NF cell proliferation assay is designed for real-time data acquisition and is well-suited for studying cell proliferation dynamics. It’s compatibility with fluorescence-based readout and the ability to customize experimental setups, CyQUANT NF assays are valuable tools for a variety of cell proliferation studies.

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Figure 4. Workflow for CyQUANT NF Cell Proliferation Assays. The CyQUANT NF Cell Proliferation assay offers a streamlined workflow that does not require freezing or cell lysis. Simply remove culture media, add the reagent containing the cell-permeant CyQUANT NF dye and plasma membrane permeabilization/dye delivery reagent, incubate, and measure the fluorescence on a microplate reader.

Figure 5. 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 graph shows the linearity that can be obtained at very low number of cells.

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CyQUANT Direct Cell Proliferation assays

CyQUANT Direct Cell Proliferation Assays offer a straightforward and efficient method for quantifying cell proliferation and determining cytotoxicity by directly staining live cells in a microplate format without the need for wash steps or fixation. Simply grow your cells in microplate wells, add the CyQUANT direct reagent and incubate, then measure the fluorescence from the bottom of the plate. The green- and red-fluorescent CyQUANT Direct dyes stain all cells, while a background suppressor blocks staining in dead or damaged cells, ensuring only live, healthy cells are stained. CyQUANT Direct assays are highly sensitive and compatible with fluorescence-based readouts, making them suitable for studying cell proliferation dynamics in various experimental settings. With their ease of use and reliable results, these assays are valuable tools for assessing cell proliferation in research and drug discovery applications.

The CyQUANT Direct Cell Proliferation Assay is a non-toxic, high-throughput screening method for continuous real-time monitoring of cell proliferation without cell lysis or fixation. The signal linearity and stability of this assay is a key feature that ensures reliable and accurate measurements of cell proliferation over time (Figure 6, 7). Further, the CyQUANT Direct assay is a valuable tool for simultaneously assessing cytotoxicity and cell proliferation dynamics in various experimental settings, enabling researchers to comprehensively analyze cell health and viability while evaluating the impact of compounds or experimental conditions on these responses (Figure 9).

The CyQUANT Direct Red Cell Proliferation Assay is a variation of the CyQUANT Direct assay that incorporates a red-fluorescent DNA-binding dye, providing the same benefits with the added advantage of red fluorescence detection (Figure 6, 8, 9), making it suitable for complex experimental setups and applications requiring multiple fluorescence readouts (Figure 10).

graphs of fluorescence vs cells per well

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Figure 6. Linearity and correlation of CyQUANT Direct assays signal. Serial dilutions of either A549 or HCASM (human coronary artery smooth muscle) cells were created. After overnight incubation, the CYQUANT Direct Red Cell Proliferation assay or CyQUANT Direct Cell Proliferation assay was used following manufacturer’s instructions. The two CyQUANT Direct assays generated similar R2 values of 0.98 and 0.99 for the A549 and HCASM cell, respectively.

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Figure 7. 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.

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Figure 8. CyQUANT Direct Red assay signal stable for 7 hours. A549 cells were seeded into a 96-well plate. After an overnight incubation, the cells were assessed using the CyQUANT Direct Red Cell Proliferation Assay following manufacturer’s instructions. The fluorescence signal was measured beginning at 5 minutes after addition of the CyQUANT Direct Red reagent until seven hours post-addition. After one hour the resulting fluorescence signal was stable up to at least seven hours.

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Figure 9. CyQUANT Direct assays produce similar screening results. A549, HepG2, and HCASM, or Jurkat cells were seeded into 96-well plates. After an overnight incubation, the cells were treated with various concentrations of tamoxifen and incubated. To determine changes in cytotoxicity, the CyQUANT Direct Red and CyQUANT Direct assays were used following manufacturer’s instructions. For each cell line the IC50 values resulting from both CyQUANT Direct assays were similar.

Figure 10. CyQUANT Direct Red assay can be multiplexed. A549 cells were seeded into the wells of a 96-well plate and incubated. After an overnight incubation, the complete medium was replaced with a staining solution containing MitoTracker Green diluted into non-serum containing medium. After a 30-minute incubation, the MitoTracker-containing medium was removed and replaced with fresh non-serum containing medium. The CyQUANT Direct Red assay was used according to manufacturer’s instructions. After a one-hour incubation, images were acquired using the EVOS FL Auto instrument equipped with the GFP (for MitoTracker Green) and the Texas Red (for detection of CyQUANT Direct Red) excitation and emission filters.