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measuring cell proliferation with SpectraMax M5, M2 and Gemini microplate readers using the CyQUANT Cell Proliferation Assay

SPECTRAMAX APPLICATION NOTE

Cathy Olsen, Ph.D., Molecular Devices Corporation, 1311 Orleans Dr., Sunnyvale, CA 94089.


INTRODUCTION
The Molecular Devices SpectraMax M5 and M2, and Gemini XPS and EM are versatile microplate readers whose detection modes include fluorescence intensity. These instruments have dual monochromators, so the optimization of excitation and emission wavelengths can easily be performed via spectral scanning without the purchase of additional individual filters. Superior sensitivity in fluorescence mode allows the user to perform a wide variety of applications with a full spectrum of fluorophores.

Quantitation of cell proliferation using fluorescence allows one to easily monitor the effects of drugs and other experimental treatments on cell growth. The CyQUANT Cell Proliferation Assay Kit from Molecular Probes is a sensitive, rapid and convenient way to quantitate cell growth using a fluorescence microplate reader. CyQUANT GR dye binds to cellular nucleic acids, allowing cell numbers to be calculated from a standard curve. Because DNA-to-RNA ratios can vary over the course of the cell cycle, the CyQUANT kit allows users to determine cell numbers using RNase-digested cell lysates and a nucleic acid standard curve.

This application note describes how to use the CyQUANT kit with the SpectraMax M5, M2 and Gemini microplate readers and SoftMax Pro software from Molecular Devices. Two methods are detailed. In the first, cellular proliferation is quantitated using a cell-based standard curve. In the second, cellular proliferation is quantitated using RNase-treated cell samples and a DNA standard curve.


MATERIALS

  • CyQUANT Cell Proliferation Assay Kit (Molecular Probes Cat. # C-7026): Component A, CyQUANT GR dye at 400X; component B, cell-lysis buffer at 20X; and component C, l DNA standard, 100 L of a 100 g/mL solution

    Note: Lysis Buffer and CyQUANT GR should be used within a few hours of being diluted. Be sure to protect the CyQUANT GR solution from light.

  • Cells: CHO-K1 cells were used to generate the data in this application note (ATCC # CCL-61)

  • EDTA if using a DNA standard curve (Fisher Chemicals Cat. # S311-100)

  • NaCl if using a DNA standard curve (Fisher Chemicals Cat. # S271-500)

  • DNase-free RNase A or RNase cocktail (Ambion Cat. # 2286)

  • Black 96-well clear-bottom microplates (Corning Cat. # 3603)

  • SpectraMax M2/M5 Multi-Detection Microplate Readers (Molecular Devices)

  • Gemini XPS/EM Microplate Fluorometers (Molecular Devices)


    CELL PREPARATION
    Adherent or non-adherent cells were prepared for the CyQUANT assay by growing and freezing them directly in the microplate(s) in which they were to be assayed, or by growing them in culture vessels, then freezing pelleted samples of the cells to be later transferred to microplates for assay. Please refer to the CyQUANT Cell Proliferation Assay Product Information sheet MP-7026 from Molecular Probes for details.


    METHOD FOR PERFORMING THE CYQUANT ASSAY USING A CELL-BASED STANDARD CURVE
    Prepare cells to be assayed for proliferation
    Step 1. Detach adherent cells from the culture plate using a trypsin or EDTA solution, and use culture medium to dilute the cell suspension into a 96-well microplate, making the cell concentrations (number of cells/well) shown in the template setup in Table 1. Include a set of control wells without cells.

    Table 1. Cell Concentration (Number Of Cells) Prepared In A Microplate Well 1 2 3 4 5 6 7 8 9 10 11 12 A 50,000 25,000 12,500 6,250 3,125 1,562 391 195 98 49 24 no cells B 50,000 25,000 12,500 6,250 3,125 1,562 391 195 98 49 24 no cells C 50,000 25,000 12,500 6,250 3,125 1,562 391 195 98 49 24 no cells D 50,000 25,000 12,500 6,250 3,125 1,562 391 195 98 49 24 no cells

    Step 2. Incubate the microplate at 37C for the desired time after which proliferation will be assayed. The incubation time will depend on the type of proliferation assay being performed, the cell type being used and the users particular experimental design.

    Step 3. Gently aspirate culture medium from wells. The wells may be washed gently with phosphate buffered saline (PBS), but this is not essential and is not recommended for very dense cultures where cells may dislodge.

    Step 4. Freeze the cells in the microplate at -70C until ready to assay (at least one hour, up to four weeks). This freezing step ensures complete lysis of cell samples.


    Prepare cells for the standard curve
    Note: It is best to use the same type of cell for the standard curve as was used in the experiment.

    Step 1. Detach adherent cells from the culture plate using trypsin or EDTA solution, and prepare a concentrated cell suspension in culture medium. Cells should be at a density of about 105 to 106 cells/mL.

    Step 2. Centrifuge 1.0 mL of the cell suspension for 5 minutes at about 200 x g (approximately 1500 rpm in a microcentrifuge). Discard the supernatant and freeze the cell pellet at -70C until ready to assay. This freezing step ensures complete lysis of the cells.


    Prepare the assay reagents
    Step 1. Dilute the concentrated cell-lysis buffer stock solution 1:20 with distilled water (prepare 200 L per well) to make 1X celllysis buffer.

    Step 2. Prepare 1X working solution of CyQUANT GR dye/cell-lysis buffer by diluting the concentrated CyQUANT GR stock solution 1:400 with 1X cell-lysis buffer. This solution should be prepared in a plastic container rather than glass (see CyQUANT product insert).


    Prepare cell-based standard curve and unknowns
    Step 1. Thaw the previously prepared frozen cell pellet at room temperature for a few minutes. Add 1.0 mL of CyQUANT GR dye/cell-lysis buffer and resuspend the cells by vortexing briefly. Assuming the pellet contained 106 cells, the resulting cell lysate contains the equivalent of 106 cells/mL.

    Step 2. Generate a dilution series in the wells of a microplate, starting at 50,000 cells/well and making serial 1:2 dilutions down to 24 cells/well. se CyQUANT GR dye/cell-lysis buffer to make dilutions such that the desired cell number is in a volume of 200 l. Prepare four replicates of each standard, and include a set of replicates without cells as a control (you can omit one of the standards, e.g., 781 cells/well, to make room on the microplate).

    Step 3. Incubate the microplate at room temperature for 2-5 minutes, protected from light.


    Set up the instrument and software
    Step 1. Turn on the SpectraMax M5, M2, or Gemini microplate reader. Launch the SoftMax Pro software and use the Assays drop-down menu to locate and open the CyQUANT Fluorescence protocol located in the Cytoproliferation folder.

    Step 2. Click the Setup button in the Plate section to launch the Instrument Settings dialogue box as shown in Table 2. After setting all of the parameters, click OK to proceed.

    Table 2. Instrument Settings Dialog
    Options
    Read Type Fluorescence
    Top read Wavelengths Ex 485
    Em 538
    Cutoff 515 Sensitivity Readings: 30
    PMT: Auto Automix Before: 5 secs AutoCalibrate On Assay Plate Type 96-well standard clear-bottom
    (default)
    96-well standard opaque Wells To Read [Determined by user] Settling Time Off AutoRead Off
    Note: Instrument settings for SpectraMax M5. SpectraMax M2 and Gemini setup options in Fluorescence mode are identical, except for the absence of the Settling Time option.

    Step 3. Create a template of the assay indicating the location of standards (cell numbers for each dilution), blanks (no cell lysate) and unknowns in the microplate. Click the Template button in the Plate section to set up the template. An example of a template with standards and unknowns is shown in Figure 1. Please note that this is the view of the template setup window that appears when the Ctrl and Shift keys are held down simultaneously to display the values assigned to each well. The assigned names of unknowns (Un01-Un12) do not appear in this view.

    Step 4. Place the microplate in the microplate reader. Use the appropriate adapter to correctly position the microplate for top reading.

    Step 5. Click the softwares Read button. The instrument will read the plate, then the relative fluorescence units (RFUs) will be displayed in the Plate section of SoftMax Pro.


    Analyze the data
    Step 1. After the microplate has been read, the data will be analyzed in the Group table that was automatically created when you set up the template.

    Step 2. In the Graph section, plot the Mean RFU Value versus Concentration (number of cells/well) from the Standards group. Configuration of the Graph Options and Edit dialog boxes is shown in Figure 2.

    Step 3. Choose the appropriate curve fit from the drop-down Curve Fit menu in the Graph sections tool bar. When plotting the standard curve for this application note, we used a log-log curve fit. (See Figure 3.) The quadratic curve fit may also be suitable.

    Step 4. Determine the number of cells per well for experimental cell samples (unknowns). The SoftMax Pro CyQUANT assay protocol calculates these values from the cell-based standard curve and lists them in the Concentration column of the Unknowns group section.


    RESULTS FOR CELL-BASED STANDARD CURVE
    A cell-based standard curve, set up as described above, is shown in Figure 3. A log-log curve fit was used when graphing the standard curve over the entire dynamic range of the assay. Results shown are from the SpectraMax M5 microplate reader, but similar results were obtained with SpectraMax M2 and Gemini EM or XPS.

    Table 3 shows sample cell proliferation assay results for CHO-K1 cells plated according to the scheme in Table 1 and allowed to proliferate in culture for two days. Cells/well on day two were calculated with SoftMax Pro using the cell-based standard curve.

    Table 3. Example Of Unknowns Group Data* Sample # Cells Plated
    Day 0
    Mean
    Cell #
    Day 2
    Std.
    Dev.
    CV% Un01 50,000 58,861 6,255 10.6 Un02 25,000 56,822 8,055 14.2 Un03 12,500 54,860 5,203 9.5 Un04 6,250 47,854 7,548 15.8 Un05 3,125 32,746 6,257 19.1 Un06 1,562 23,036 4,533 19.7 Un07 391 9,262 2,884 31.1 Un08 195 5,198 1,873 36.0 Un09 98 3,856 714 18.5 Un10 49 2,690 814 30.3 Un11 24 2,208 179 8.1

    * Mean concentration (cells/well) of cells two days after plating is calculated.


    METHOD FOR PERFORMING THE CYQUANT ASSAY USING A DNA-BASED STANDARD CURVE AND RNASE-TREATED CELLS
    Cell proliferation can also be assayed based on DNA content using a DNA standard curve. The standard curve can serve to quantitate cellular DNA, provided the cell lysates are pretreated with DNase-free RNase to prevent the RNA from contributing to the fluorescent signal. In this application note, instructions are provided for setting up the DNA standard curve and RNase-treated cell samples.


    Prepare the assay reagents
    Step 1. Dilute the concentrated cell-lysis buffer stock solution 1:20 with distilled water (prepare 200 L per well) to make 1X lysis buffer work solution. Prepare two types of 1X lysis buffer: one with a final concentration of 1mM EDTA and 180 mM NaCl and one without EDTA or NaCl.

    Step 2. Prepare 1X and 2X working solutions (100 L per well) of CyQUANT GR dye/ cell-lysis buffer by diluting the concentrated CyQUANT GR stock solution 1:400 or 1:200, respectively, with working strength cell-lysis buffer. This solution should be prepared in a plastic container rather than glass, as recommended by Molecular Probes.


    Prepare a DNA standard in plastic tubes:
    Step 1. Dilute the 100 g/mL DNA standard from the kit in 1X CyQUANT GR dye/celllysis buffer to make a 1 g/mL stock solution.

    Step 2. Dilute the 1 g/mL stock solution further to make a dilution series as shown in Table 4.

    Table 4: Preparation Of Dilutions For DNA Standard Curve For The CyQUANT Cell Proliferation Assay Tube
    #v
    1x CQ GR/
    Lysis
    Buffer (L)
    1 g/mL DNA in
    1x CQ GR/Lysis
    Buffer (L)
    Final DNA
    Concentration
    (ng/mL)
    1 1000 0 0 2 990 10 10 3 950 50 50 4 900 100 100 5 800 200 200 6 600 400 400 7 400 600 600 8 200 800 800 9 0 1000 1000

    Note: The CyQUANT kit includes a l bacteriophage DNA standard. The data shown in this application note were generated using this standard, prepared in serial dilutions. Depending upon the assay you are running, you may wish to use the kits DNA standard or another DNA that is more appropriate for your sample, prepared according to the instructions in the Molecular Probes Product Information sheet MP-7026.


    Prepare the cells
    Choose the appropriate set of culture and freezing instructions from Molecular Probes CyQUANT Product Information sheet for the type of cells (adherent or non-adherent) and growing conditions (in microplates or culture vessels). Freeze the cells at -70C. For this application note, adherent cells were trypsinized to detach them from the culture vessel, then pelleted and frozen. Samples containing known numbers of cells were used to demonstrate assay performance.


    Prepare the cell samples, standard curve DNA samples and blanks
    Step 1. Remove the previously prepared frozen cell pellets (or microplate) from -70C storage. Thaw the frozen cells at room temperature for a few minutes.

    Step 2. Pre-treat the cells with RNase by adding 100 L 1X cell-lysis buffer containing 180 mM NaCl and 1 mM EDTA to each well and to four wells without cells (blanks). If using previously pelleted cells, resuspend each sample in 100 L and transfer to the wells of a microplate. Add 4 L of RNase (two units per well; see note below) into each well containing cells as well as the control wells without cells. Incubate at room temperature for one hour.

    Note: DNase-free RNase should be used at a concentration such that no more than 10 L contains the full number of units needed to treat a sample. For example, the RNase Cocktail from Ambion has 500 U/ mL activity, so 4 L per well equals two units per well.

    Step 3. Add 100 L of 2X CyQUANT GR dye/ cell-lysis buffer to each of the microplate wells, including the control wells without cells.

    Step 4. Prepare the DNA standard curve by transferring the previously prepared DNA serial dilutions into the appropriate wells of the microplate, as defined in the template editor. The plate should contain two controls: a digest control prepared following the same steps as the cellular samples, but without cells (no DNA), and a standard curve control/plate blank containing only 200 L 1X CyQUANT GR dye/cell-lysis buffer (no DNA and no RNase).

    Step 5. Incubate the samples for 2-5 minutes.


    Set up the instrument and software
    Step 1. Turn on the microplate reader. Launch the SoftMax Pro software and use the Assays drop-down menu to locate and open the Basic endpoint protocol in the Basic protocols folder.

    Step 2. Set up the Instrument Settings dialog box as shown in Table 2.

    Step 3. Create a template of the assay showing where standards, unknowns (cells treated with RNase), and plate blanks will be located on the microplate. To access the Template Editor, click the Template button in the tool bar of the Plate section.


    Read the plate and analyze the data
    Step 1. Place the microplate in the reader.

    Step 2. Read the plate. RFUs will be displayed in the Plate section.

    Step 3. After the microplate has been read, the RFUs will be displayed in the Plate section. The data will be analyzed in the Group tables that you created while setting up the template.

    Step 4. Plot the mean RFU of the Standards group versus the concentration of the Standards group in the Graph section for the DNA standard curve.

    Step 5. Choose the appropriate curve fit from the drop-down Curve Fit menu in the Graph sections tool bar. We used a log-log curve fit for this experiment.

    Step 6. Calculate the cellular DNA values in RNase-treated cells from the DNA standard curve using the built-in formula in SoftMax Pro. An example of the Graph Options and Edit dialogue boxes are shown in Figure 2.


    RESULTS FOR DNA STANDARD CURVE
    A typical DNA-based standard curve is shown in Figure 4. The SpectraMax M5 microplate reader was used to generate these data, but similar results were obtained on the SpectraMax M2 and Gemini microplate readers (data not shown). An example of the calculation of cellular DNA concentrations in RNase-treated cells from a DNA standard curve is shown in Table 5. For this application note, a serial cell dilution was used as unknowns. In fact, any number of cells between 50 and 50,000 cells/well (in the linear range of the standard curve) can be detected and cellular DNA value can be obtained using this method.


    Table 5. Sample Group Table: Cellular DNA Concentrations Calculated By SoftMax Pro Software Using DNA Standard Curve* Cells/ Well Mean RFU Mean Cell DNA (ng) Std. Dev. Cell DNA CV (%) 50,000 554.8 40.29 1.20 3 25,000 375.6 26.40 0.40 1.6 12,500 195.8 13.04 0.46 3.5 6,250 106.1 6.71 0.34 5 3,125 58.5 3.52 0.24 6.8 1,563 30.7 1.75 0.12 6.7 781 17.0 0.93 0.16 17.4 391 7.1 0.36 0.08 21.8 195 6.2 0.31 0.05 14.9 98 3.0 0.14 0.04 27.8 49 2.7 0.13 0.03 20.8

    * Replicates are not presented in this view of the group table.

    Figure 5 shows the average cellular DNA in each set of samples versus the number of cells/well. The linear range of the assay under these conditions is from 50 to 50,000 cells, as expected based on Molecular Probes Product Information sheet MP 7026. A comparison of RFUs in RNase-treated and untreated cell samples is shown in Figure 6 and demonstrates that RNase treatment reduces the amount of fluorescence in the cell lysates.


    CONCLUSIONS
    The CyQUANT Cell Proliferation Assay is a quick, sensitive fluorescence method for quantitating cell numbers or cellular DNA content. Assay results are similar on the SpectraMax M5 or M2, and Gemini XPS or EM microplate readers. Using a cell-based standard curve and a five-minute incubation, we observed a limit of detection and dynamic range similar to that described in Molecular Probes CyQUANT Product Information sheet (50-50,000 cells per sample). Using a DNA standard curve generated with the l DNA standard provided with the kit, we were able to calculate the amount of DNA in as few as 50 cells. The analysis capabilities of SoftMax Pro software provide a flexible, convenient method for calculating and reporting the data.


    REFERENCES
    1. Molecular Probes Product Information sheet MP 7026 1/12/01: CyQUANT Cell Proliferation Assay Kit (C-7026).


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