Youssef Farhat, Written 7/16/12, Last updated 5/6/14
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Introduction
The PicoGreen assay is useful for quantifying the amount of double-stranded DNA (dsDNA) that is present in a given sample. The measured amount of dsDNA can then be used as a marker for cell proliferation.
To use this assay, samples must first be thoroughly homogenized. The protocol below uses the protease, Papain, and an overnight incubation at 65ºC in order to dissolve the samples and completely release their dsDNA into solution.
The samples used in this protocol were ~400 µL collagen gels seeded with approximately 300,000 cells. The samples had been collected into 1.5 mL Eppendorf tubes and frozen at -80°C immediately after harvesting until the day they were processed for this assay.
Materials
- Quant-iT™ PicoGreen ® dsDNA Kit (Invitrogen, #P11496). This kit contains
- Quant-iT™ PicoGreen ® dsDNA Reagent (10 vials, each containing 100 µL aliquots)
- 25 mL of 20X TE buffer
- 1 mL of 100 µg/mL Lambda DNA (dsDNA standard)
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- Papain (Sigma-Aldrich, #P4762-25MG)
- L-cysteine (Sigma-Aldrich, #C1276-10G)
- EDTA (Sigma-Aldrich, #E6511)
- DPBS (Gibco, #14190)
- Ultrapure distilled water (Gibco, #10977)
- Black, flat-bottomed 96-well plates (e.g. Dynex, #3010 or Greiner Cellstar, #655086)
- An oven set to 65ºC
- A plate reader
- Typical lab equipment and supplies such as pipettors, micropipettors, pipette tips, autoclaved bottles, Eppendorf tubes
Procedure
1. Prepare and aliquot reagents
1) Prepare Papain stock solution (10 mg/mL) by dissolving the entire bottle of Papain (containing 25 mg) powder into 2.5 mL of DPBS. Aliquot a combination of 100-300 µL into Eppendorf tubes and store them at -20°C.
2) Prepare dsDNA standard aliquots by adding 8 µL of the provided standard (100 µg/mL) into several Eppendorf tubes. Store at -20°C.
3) Prepare L-cysteine stock solution (100X, at 24.2 mg/mL) by measuring 242 mg of L-cysteine and dissolving it into 10 mL of DI water. Aliquot 0.5 mL into Eppendorf tubes and store at -20°C.
4) Prepare EDTA stock solution (0.333 M) by combining 13.859 g of EDTA powder with 100 mL of deionized water. Store at 4°C for up to 3 months.
5) Prepare enough Papain digestion solution for the number of samples to be processed. In this protocol, 1 mL of digestion solution is needed per sample. To make 50 mL of digestion solution, combine the following into a clean bottle:
- 48.6 mL of DPBS
- 625 µL of Papain stock solution (final concentration of 125 µg/mL)
- 500 µL of L-cysteine stock solution (final concentration of 0.242 mg/mL, or 2 mM)
- 300.3 µL of EDTA stock (0.333 M)
2. Digest samples with Papain and equalize their volumes for the assay
1) Remove samples from the freezer (if frozen) and add 1 mL of digestion solution to each sample.
2) Vortex samples until they begin to float in the digestion solution (about 15 seconds).
3) Place samples in the 65°C oven overnight to digest completely.
The next day…
1) Remove the samples from the oven and allow them to cool until they can be handled comfortably.
2) Lightly vortex the samples to suspend any remaining particles that may have settled and return them to the oven for 30 min.
3) Remove the samples from the oven and allow them to cool until they can be handled comfortably.
4) Weigh each sample tube and equalize their volumes with deionized water to account for variations in sample volume.
5) Lightly centrifuge the samples to settle any remaining particles to the bottom of the tube (lowest setting for 1 min).
3. Perform the PicoGreen Assay
1) Prepare 40 mL of 1X TE buffer by adding 2 mL of 20X TE buffer to 38 mL of ultrapure distilled water.
2) Dilute enough PicoGreen reagent for the assay 200 times in 1X TE buffer. 100 µL of diluted reagent is needed per measurement. So, for a 96-well plate, 10 mL can be prepared by combining 50 µL of reagent with 10 mL of 1X TE buffer. Protect the reagent from light and use the solution within a few hours of preparing it.
3) Prepare the dsDNA standard curve by first diluting 8 µL of dsDNA standard aliquot in 392 µL of 1X TE buffer (final concentration is 2 µg/mL). Then, perform 1:1 serial dilutions with 1X TE buffer in cDNA tubes until the 7th dilution. Leave the 8th dilution pure 1X TE buffer to serve as a blank.
4) Prepare the samples by diluting at least 10 times in 1X TE buffer. This will reduce the interference of the Papain digestion solution or other agents in the samples with the assay. (You can verify whether this step is necessary by taking a single sample and serially diluting it in 1X TE buffer. Observe how many dilutions are necessary before the measured fluorescence vs. the concentration of each dilution becomes a straight line).
5) Add 100 µL of each sample and standard to the black, 96-well plate.
6) Add 100 µL of the diluted PicoGreen reagent into each well of the plate.
7) Incubate the samples at room temperature for 2-5 minutes while protecting them from light. Read the fluorescence of each well by setting the plate reader to an excitation wavelength of 480 nm and emission wavelength of 520 nm.
Citing this Protocol
Farhat, Y. “PicoGreen Cell Proliferation Assay Protocol.” The Protocol Place. July 2012. Accessed on mm/dd/yyyy. <http://protocol-place.com>
