Section12:Setting Up a Cell Proliferation Assay

From Assay Guidance Wiki

Obtain a minimum of two vials of cells from ATCC, store 1 vial in liquid nitrogen and thaw out the other vial. For cell lines that are unavailable from ATCC and were developed internally, obtain the lowest passage cell stock. Culture cells in recommended media for two to three passages allowing the cells to reach no more than 60-80% confluence. Expand cells in a couple T150 flasks and freeze. Conduct a test for mycoplasma contamination on a aliquot using a commercial kit every time new cells are frozen for storage.

It is best to freeze for future use at least 10-20 vials of cells at 1 – 5 x 106 cells/0.5ml of culture media that contains 10% DMSO or other additive, as recommended by ATCC, since some cells can not be frozen with DMSO. For nonadherent cells, such as T and B cell hybridomas, which lose chromosomes and generate variants easily, the maximum cell density should be 7 x 105 cells/ml. Cells should not be used after 10 to 20 passages or passaged for more than two to three months to limit the number of doublings the cells undergo, as cell lines are genetically unstable and will change with multiple doublings. Some cell lines may need tighter limits on passage number, which should determined empirically by testing the length of time the desired sensitivity or response can be obtained. These studies are usually done in parallel by pulling out a new ampoule in case sensitivity is lost. Low passage numbers help assure the least amount of genetic modification has occurred and that results are reproducible from one experiment to another.

Determine the Doubling Time of the Cells

In T-25 tissue culture flasks, seed 5,000 cells per flask. Count the cells each day at the same time for up to 7 days using a hemacytometer after staining with Trypan Blue. It is best to set up duplicate flasks for each day.

Procedure for determining population doubling times for adherent cells.

On day 0, prepare 10 T25 flasks, each containing 10 ml of cells at 1.25 x 104/ml (this will give 5000 cells/cm2). Use standard growth medium for cell line. Place flasks in incubator.
For the following 5 days at 24 hour intervals in duplicate, draw off all of the media with a pipet, add 0.5 ml of trypsin, manipulate the flask until the entire cell growing surface of the flask is covered with trypsin.
Incubate the flask at 37C until the cells are completely detached (observe under the microscope to confirm that the cells are detached).
Add 5 ml of media to flask, mix to suspend cells, transfer to conical tube, and centrifuge.
Remove media and add 1.0 ml of trypan blue.to pellet. Resuspend with vortex.
Immediately count the viable cells (cells that do not take up the blue color) on a hemacytometer.
Plot log of total viable cells vs time in hours.
Select the linear portion of the growth curve for linear regression analysis. The doubling time is obtained by dividing 0.3 by the slope of the line.

Cells should ALWAYS be examined visually before and after working with them to make sure they look healthy and no obvious harm has come to them.

Determine Linear Correlation of Proliferation Detection Reagent with Cell Number

For the proportionality test, it is best to use the range of cell numbers found to be in the linear range from the previous doubling time determination experiment. The table above compares the reagents commonly used to follow cell proliferation and describes the advantages and disadvantages of each method.

Prepare a cell suspension in culture media and perform a cell count on an aliquot using a hemacytometer after staining with Trypan blue. A Coulter Counter, which counts particles and does not distinguish between live and dead cells, is not recommended. However, if a Coulter Counter is used, the results obtained need to be checked frequently with Trypan blue staining to verify the Coulter counter results.

Plate varying number of cells per well in 96-well microtiter plates (start out with 5,000 cells/cm2 or higher for very small cells followed by 1:2 fold serial dilutions). Allow the cells to adhere or settle for at least 4 hours at 37°C. Add the proliferation detection reagents to wells and process according to manufacturer’s instructions. The incubation time of the proliferation detection reagent may need to be optimized if the manufacturer’s recommendation does not give a suitable response. The optimal incubation time should be used in all subsequent experiments. Care should be taken not to exceed the optimal range of the detection system and to make sure the reader is working properly, which should be checked with standard plates if an absorbance greater than 1.0 needs to be used.

Determine the linearity of the proliferation assay with cell number by plotting response vs. cell number and analyzing by a weighted linear regression analysis, where the weights are the reciprocals of the observed values. When the response is proportional to the cell number, the value predicted from the fitted line at each cell density should be two times the predicted value of the next lower cell density (assuming 1:2 fold dilutions). For each cell density above the lowest one, the ratio of the predicted value to the previous predicted value is calculated and divided by 2. If 1:2 fold dilutions were not used, the ratios of the corresponding cell densities should be calculated and used instead of 2. These ratios are then plotted and will be between 80% and 120% of the expected signal when the signal is proportional to the cell number. The signal is only proportional to the cell number for the of range cell densities that pass this test. All subsequent experiments should use cell densities that will ensure that results are within the linear/proportional range and do not exceed the upper or lower limits of this range.

In general, DMSO is the preferred solvent for dissolving compounds used in in vitro studies or assays. A DMSO tolerance study should be performed to determine the maximum amount of DMSO added to media that does not compromise the health of cells over two to three doublings, as measured above. The maximum amount of DMSO is usually 0.5-1% but may vary with the cell type.

The DMSO concentration to be used in the cellular assay is dependent upon the results of DMSO tolerance experiments and the highest concentration of compound to be tested. The media used to generate serial dilutions of compounds should contain the same percentage of DMSO that is in the highest concentration of compound. For example, let's suppose the highest compound concentration to be tested is 100 M. From a 20 mM stock in DMSO, one first makes a 1:20 dilution of the compound in culture media (DMSO concentration at this step is 5%). Ten microliters of the 1:20 dilution of compound is then added to 90 l of cells (final DMSO concentration is 0.5%). Therefore, a 5% DMSO in culture media should be used to make the serial-fold dilutions of the compound which are then added to the cells to give a final DMSO concentration of 0.5%.

Determine Optimum Initial Cell Density

Plate various cell densities based on the results obtained in step 3 (generally between 500 and 10,000 cells/well), in 96 well plates using an experimental design template that will allow for the analysis of positional effects. The template consists of instructions on setting up serial dilutions of cells in plates in the presence and absence of a standard inhibitor. Multiple plates are prepared and incubated for 48, 72, and 96 hours (an additional fourth incubation time of 120 hours is used for slow growing cells). The inhibitor should be chosen to match the individual target being studied. For instance, a cell cycle inhibitor like vineomycin would be best for studying cell cycle inhibition, while staurosporine would be better for studying kinase inhibitors and vinblastine as a cytotoxic agent. The DMSO concentration must be held constant as the inhibitor concentration is varied, by preparing serial dilutions of the compounds as described above. The concentration ranges for the inhibitors should bracket the IC50 for inhibition with half the points being above and half below the IC50 value.

Cells in the plates are grown for 16 hours before adding inhibitor so as to allow the cells to adhere to the wells and start growing. Cells should be treated with inhibitor for two doubling times but no longer than three doubling times. The order of the serially diluted cells outlined in the template, will allow for the simultaneous assessment of edge effects, column drift and optimal incubation time. The template will automatically carry out the statistical analysis of drift effects, edge effects, and optimal incubation times. A statistician can review a copy of the template and the completed analysis to verify its interpretation. Template to Optimize Conditions

Mini-validation Study

Once the optimal initial cell density has been determined, a mini-validation study will determine the signal window and signal to noise ratio. A mini-validation study is also performed when transferring a proliferation assay from one lab to another lab (see results in link above to use as starting conditions). These validation studies are described in the Assay Gidance Manual and involve using two plates on each of two separate days, in which one-third of the wells contain the recommend initial cell density, another third contains no cells, and the remaining third contain a control inhibitor or some other condition that achieves a mid-level signal. The wells containing media alone are used to subtract a baseline value from the experimental data points, while cells treated with high concentrations of a cytotoxic agent (>95% cell kill) represents the minimum signal. Untreated cells represent the maximum signal.

An experimental design is recommended if the validation study does not yield a suitable signal window and signal to background ratio. Consult a statistician to carry out an experimental design study to optimize the variables that impact the assay.

IC50 Determinations

Eight to ten inhibitor concentrations are required for IC50 determinations using non-linear regression analysis, which allows for a concentration range to be studied that includes concentrations above, and below the IC50 value.

Test-Retest

For in vitro assays, a within-run minimum significant ratio (MSR) of 3 or less is required for a validated assay. An MSR between 3 and 4 may be acceptable if the assay controls from production runs are closely monitored (consult a statistician). In general the MSR calculation requires 20 - 30 or more IC50 determinations run twice, as described in the LOB handbook. It is recommended that the CV of the IC50 for individual curve fits be less than or equal to 40%, which corresponds to an MSR of 3.

Outliers from the IC50 curve can be determined by calculating the IC50 with and without the suspected data point. If the IC50 values are within the msr, then the point is most likely not an outlier and should be included in the calculation. In general statistical tests for outliers do not perform well in small samples (< 20). When deciding if a point is an outlier, the following rule of thumb is recommended: “If it is not an obvious outlier, it is not an outlier.”