Cell proliferation analyses are crucial for cell growth and differentiation studies, and are often used to evaluate both compound toxicity and inhibition of tumor cell growth during drug development. Proliferation measurements are typically based on average DNA content or cellular metabolism, or they quantify DNA synthesis. Our broad range of Molecular Probes® cell analysis assays can be used individually or together to carry out complex biological investigations, including studies of cell proliferation, cytotoxicity, or drug efficacy, in microplate assays or using imaging or flow cytometry platforms.

DNA content

CyQUANT® cell proliferation assays provide an accurate microplate-based fluorescence method for counting cells in a population, based on cellular DNA content. Because cellular DNA content is highly regulated, 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.

CyQUANT® Cell Proliferation Assay Kit
Quantitation of NIH 3T3 fibroblasts using the CyQUANT® Cell Proliferation Assay Kit.

New DNA synthesis—imaging/high-content analysis

Measuring the synthesis of new DNA is a precise way to assay cell proliferation in individual cells or in cell populations. The Click-iT® EdU assay measures the rate of new DNA synthesis based on incorporation of the nucleoside analog EdU into DNA. Detection is achieved through a copper-catalyzed “click” reaction that is typically completed within 30 minutes.

Click-iT® Plus EdU Imaging Assays use mild reaction conditions and offer a range of fluorescence options, making these reagents multiplex-compatible for content-rich results in microplate assays. In addition, the Click-iT® EdU Microplate Assay is optimized for microplate-based fluorescence analysis with signal amplification and a simple and rapid workflow for use in HTS.

Selection guides
  CyQUANT® Cell Proliferation Assay Kit CyQUANT® NF Cell Proliferation Assay Kit CyQUANT® Direct Cell Proliferation Assay
Target DNA content DNA content DNA content
Reporter CyQUANT® GR CyQUANT® NF CyQUANT® Direct
Ex/Em (nm) 480/520 480/520 480/520
Sample Cultured cells must be frozen during multi-point tests and assayed together with common standards Streamlined protocol for fast readout; eliminates freezing step; 1-hour assay from start to finish 15-minute protocol; no freezing, lysis, or temperature equilibration; viability indicator (i.e., does not count dead cells)
Usage Detects in the range 50–50,000 cells Detects in the range 100–20,000 cells Detects in the range 50–20,000 cells
Protocol outline
  1. Prepare reagent.
  2. Dispense cells into plate wells.
  3. Incubate 4–6 hours to allow cells to attach.
  4. Remove media and freeze at –70°C.
  5. Thaw plate, add reagent.
  6. Incubate for 2–5 min at room temperature.
  7. Measure fluorescence.
  1. Plate and grow cells overnight.
  2. Remove culture media.
  3. Add reagent, incubate 30–60 min.
  4. Measure fluorescence.
  1. Plate and grow cells overnight.
  2. Remove culture media.
  3. Add reagent and incubate 30–60 min.
  4. Measure fluorescence.
Components All assay components All assay components All assay components
Format 1 kit, 1,000 assays 1 kit, 200 assays 1 kit, 10 plates
Cat. No. C7026 C35006 C35011
  Click-iT® EdU Microplate Assay
Target The modified thymidine analogue EdU is incorporated into newly synthesized DNA in proliferating cells.
Reporter Amplex® UltraRed-amplified Click-iT® EdU reaction
Ex/Em (nm) 571/585
Sample Optimized for measuring proliferation in live cell populations, click detection step comes after fixation.
Usage Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Components All assay components
Format 400 assays
Protocol outline
  1. Add EdU to plated cells.
  2. Remove media, add fixative, incubate 5 min.
  3. Add prepared Click-iT® reagent, incubate 25 min.
  4. Remove prepared reagent, wash 2X.
  5. Add HRP-conjugated antibody reagent.
  6. Remove antibody reagent, wash, add Amplex® UltraRed buffer, incubate 15 min.
  7. Add stop reagent, read fluorescence.
Cat. No. C10214
For Research Use Only. Not for use in diagnostic procedures.