Protocol for CyQUANT™ Cell Proliferation Assay

1. Introduction

The Turner BioSystems TD-700 Laboratory Fluorometer in combination with Molecular Probes' CyQUANT^TM Cell Proliferation Assay Kit provides a convenient, rapid and sensitive procedure for determining the density of cells in culture. The assay has a linear detection range extending from 50 or fewer to at least 200,000 cells in 2 mL volumes (Figure 1) and is therefore ideal for cell proliferation studies as well as for routine cell counts. The assay is based on dye fluorescence enhancement upon binding to cellular nucleic acids. (1) Cells are simply lysed by addition of a buffer containing the CyQUANT-GR dye; there are no washing steps, growth medium changes or long incubations. The resulting fluorescence is proportional to the number of cells in the sample and is measured directly using the TD-700 fluorometer equipped with a fluorescein filter kit. The CyQUANT assay can detect much lower cell numbers than Neutral Red or methylene blue assays. (2,3,4) Unlike procedures that rely on the conversion of tetrazolium dyes to blue formazan (5) products or 3H thymidine incorporation assays, (6) the CyQUANT method is rapid and does not rely on cellular metabolic activity. Thus, cells can be frozen prior to assaying; time course assays are facile and data obtained from samples taken at widely different time intervals can be directly compared. Also, unlike tetrazolium conversion assays, serum components do not appreciably interfere with the assay. The CyQUANT assay performs reliably with widely disparate cell types, including mouse fibroblasts (NIH 3T3 and CREBAG 2 cells), normal human umbilical vein endothelial cells (HUVEC, InvitroCyte, Inc., Seattle), canine kidney cells (MDCK), chinook salmon embryo cells (CHSE), rat basophilic leukemia (RBL), rat glioma cells (C6) and mouse myeloma cells (P3X63A68).

2. Materials Required

• TD-700 Fluorometer with standard PMT and 10 mm x 10 mm square cuvette adaptor (P/N 7000-009).
• Blue Mercury Vapor Lamp (P/N 10-089) or Quartz-halogen lamp (P/N 7000-930).
• Fluorescein filter kit (P/N 10-086R).
• 10mm x 10 mm square methacrylate disposable cuvettes (P/N 7000-959).
• CyQUANT Cell Proliferation Assay Kit (catalog number C-7026), supplied by Molecular Probes, Inc., Eugene, OR. The kit contains 550µL of 400X CyQUANT-GR stock solution in DMSO, 11 mL of 20X Cell Lysis Buffer, and 100µL of 100µg/mL lamda -DNA Standard in TE buffer (10 mM Tris, 1 mM EDTA). The kit contents are sufficient for 100 assays using 2.0 mL samples in 10 mm x 10 mm cuvettes. Handling, storage and the use of the reagents should be performed in accordance with the product information sheet supplied by Molecular Probes, Inc.

3. Experiment Protocol

3.1 Reagent Preparation

On the day of the experiment, dilute the concentrated Cell Lysis Buffer stock solution 20-fold in distilled water (2.0 mL will be required for each assay). Just prior to running the experiment, dilute the CyQUANT-GR stock solution 400-fold into the 1X Cell Lysis Buffer. For example, to prepare 20 mL of CyQUANT-GR working solution (enough for ~10 assays), first make the 1X Cell Lysis Buffer by mixing 1 mL of the 20X stock with 19 mL of nuclease-free distilled water; next add 50µL of the CyQUANT-GR stock solution and mix thoroughly. We recommend preparing the working solution in a plastic container, rather than in glass; the CyQUANT-GR reagent may adsorb to glass surfaces. Protect the working solution from light by keeping it in an opaque bottle, covering it with foil or placing it in the dark to prevent photodegradation of the CyQUANT-GR dye. For best results, the solution should be used within a few hours of its preparation.

3.2 Cell-Number Standard Curve

A reference standard curve can be created for converting sample fluorescence values into cell numbers. The cell type used for the standard curve should be the same as that which is used in the experiment. It is possible to assay either suspension cells or adherent cells, however, the latter must first be detached and suspended by treatment with trypsin. Note that some adherent cells are sensitive to trypsinization and some cell lysis might ensue.

3.2.1 Prepare a concentrated cell suspension in medium: ideally this should be ~1 mL total volume at a density of about 5 x 10⁵ - 1 x 10⁶ cells/mL. Determine the actual cell density by counting the cells using a hemacytometer(7) or optical density measurements.(8)

3.2.2 Centrifuge 1.0 mL of the concentrated cell suspension for 5 minutes at 200 X g (1500 rpm in a microcentrifuge). Carefully remove and discard the supernatant without disturbing the cell pellet, and freeze the cell pellet at -70EC (note [A]).

3.2.3 Thaw the cell pellet at room temperature, add 1.0 mL of the CyQUANT-GR dye/Cell Lysis Buffer (prepared in Section 3.1), and resuspend the cells by briefly vortexing.

3.2.4 In a set of culture tubes, serially dilute the cell suspension with CyQUANT-GR dye/Cell Lysis Buffer to obtain a range of 2.0 mL samples containing numbers of cells from 50 to 200,000. Include a 2.0 mL sample with no cells as a reagent blank. Incubate for 2-5 minutes at room temperature, protected from light.

3.2.5 Transfer samples to standard acrylic fluorescence cuvettes and measure the fluorescence using the TD-700 fluorometer with the fluorescein filter set (P/N 10-086R). Insert the most fluorescent sample first and calibrate the instrument sensitivity as directed in the TD-700 manual (press #2, calibrate). This procedure automatically optimizes the instrument sensitivity to match the fluorescence of the sample.

3.2.6 Measure the fluorescence of the remaining samples. To equalize any photobleaching effects, insert samples into the fluorometer for approximately equal time periods. The fluorescence value of the reagent blank (CyQUANT-GR dye + Cell Lysis Buffer only) may be subtracted from that of each sample. Corrected or uncorrected data may be used to generate a standard curve of fluorescence versus cell number (for example, see Figure 1). The form of the standard curve will vary with cell type.

Figure 1. Quantitation of MDCK (Madin-Darby canine kidney) cells using the CyQUANT Cell Proliferation Assay Kit and the TD-700 fluorometer. Upper panel: 1000 to 200,000 cells per 2.0 mL sample. Lower panel: 50 to 1000 cells per 2.0 mL sample.

3.3 Cell-Number Determination: Cells Grown in Standard Culture Conditions

The CyQUANT Cell Proliferation Assay Kit can be used to count the number of cells in a sample taken from a conventional cell culture. Adherent cells must be first detached by treatment with trypsin and then suspended. Note that some lysis may occur in adherent cells that are sensitive to trypsinization. Cells grown in suspension can be assayed directly.

3.3.1 Transfer suspended cell samples to centrifuge tubes and centrifuge for 5 minutes at 200 X g (e.g., 1500 rpm in a microcentrifuge). Samples should contain from about 50 to about 200,000 cells. Remove and discard the supernatant without disturbing the cell pellet, and freeze the cell pellet at -70EC (note [A]).

3.3.2 Thaw the cell pellets at room temperature and add 2.0 mL of CyQUANT-GR/Cell Lysis Buffer (prepared in Section 3.1) to each sample.

3.3.3 Transfer entire 2.0 mL samples to acrylic fluorescence cuvettes and measure the fluorescence using the TD-700 fluorometer using the same instrument parameters as used in generating the standard curve (Section 3.2.5). To equalize any photobleaching effects, insert samples into the fluorometer for similar time periods to those used for the standard curve measurements.

3.3.4 If the standard curve was plotted using blank-subtracted data (Section 3.2.5), the reagent blank (CyQUANT-GR dye + Cell Lysis Buffer only) fluorescence value must also be subtracted from that of each of the samples. Convert the observed fluorescence to cell number using a standard curve (Section 3.2.6). We have found that many different cell types can be assayed using this procedure, but the absolute signal is cell type-dependent. Thus it is advisable to use a standard curve generated from the same cell type that is being assayed, for comparison. Alternatively, a standard curve generated using pure DNA (Section 3.5) can be calibrated relative to an appropriate cell type.

3.4 Cell Number Determination Based on DNA or RNA Alone

In the protocols described above, the CyQUANT-GR reagent is used to determine cell number by staining both DNA and RNA. DNA to RNA ratios, however, may vary according to cell type and cell cycle position. Fluorescence due to CyQUANT-GR dye binding to RNA can be eliminated by pretreating samples with DNase-free RNase. Likewise, fluorescence due to DNA-bound dye can be eliminated by pretreating samples with RNase-free DNase.

3.4.1 For determination of total cellular DNA or RNA, freeze a cell pellet containing 20,000-100,000 cells at -70EC, thaw at room temperature, and resuspend in 1 mL of 1X Cell Lysis Buffer, containing 180 mM NaCl. For RNase treatment, this buffer should also contain 1 mM EDTA. For DNase treatment the buffer should contain 1 mM CaCl2 and 1 mM MgCl2.

3.4.2 DNase-free-RNase A or RNase-free-DNase I is added to a final concentration of about 1.35 Kunitz units/mL (RNase) or 45 Kunitz units/mL (DNase). (9,10) Samples are incubated for one hour at room temperature.

3.4.3 An equal volume of a 2X working solution of CyQUANT-GR dye (50µL of the CyQUANT-GR/DMSO stock solution per 10 mL of 1X Cell Lysis Buffer) is added to each sample. Samples are incubated for 2-5 minutes.

3.4.4 The fluorescence is measured as described above (3.2.5). It is suggested that controls be run for each digested sample, using the appropriate buffer, as the presence of salt and divalent cations slightly reduces the slope of the standard curve.

3.5 DNA Standard Curve

The CyQUANT Cell Proliferation Assay Kit includes a 100µg/mL sample of bacteriophage lambda-DNA that can be used to prepare a standard curve for DNA content. The standard curve can serve to quantitate cellular DNA, provided that the cell lysates are pretreated with DNase-free RNase to eliminate the RNA component of the fluorescence signal (Section 3.4, above). Alternatively, the standard curve can be used to calibrate the assay for use of the fluorometer at different times or on different days. Variation in the signal intensity of the standard curve is directly related to variation that will be observed for assaying cells on different days, and is instrument-dependent. In a set of culture tubes, prepare serially diluted 2.0 mL samples of bacteriophage lamda-DNA with concentrations ranging from 50 pg/mL to 0.5µg/mL by diluting the 100µg/mL stock solution into CyQUANT-GR/Lysis Buffer working solution (as prepared in Section 3.1). Include a reagent blank sample without DNA. Measure fluorescence as described above (3.2.5 and 3.2.6).

Note
[A] Frozen cell pellets in centrifuge tubes can be stored at -70° C for up to four weeks. The freezing step is important for efficient cell lysis in the CyQUANT assay.

4. References

1. J Immunol Meth 142, 199 © 1991
2. In Vitro Toxicology 3, 219 © 1990
3. Biotechnic and Histochemistry 68, 29 © 1993
4. Anal Biochem 213, 426 © 1993
5. Cancer Res 48, 4827 © 1988
6. Exp Cell Res 124, 329 © 1979
7. Meth Enzymol 58, 141 © 1979
8. BioTechniques 21, 260 © 1996
9. J Gen Physiol 24, 15 © 1940
10. J Gen Physiol 33, 349 © 1950

5. Patent and Trademark Information

CyQUANT is a trademark of Molecular Probes, Inc. Materials and methods incorporated in the CyQUANT Cell Proliferation Assay Kit are covered by current or pending U.S. and foreign patents.